Particulate drug delivery methods

ABSTRACT

The invention provides compounds and compositions useful for the modulation of certain enzymes. The compounds and compositions can induce of cell death, particularly cancer cell death. The invention also provides methods for the synthesis and use of the compounds and compositions, including the use of compounds and compositions in therapy for the treatment of cancer and selective induction of apoptosis in cells.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/679,129, filed Aug. 3, 2012, whichis incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Apoptosis is a process used by higher organisms to maintain homeostasisby removing cells that are in excess, damaged, or potentially dangerous.Caspase enzymes are a class of cysteine proteases that cleave cellularsubstrates after recognition sequences with C-terminal aspartateresidues. The activation of caspase enzymes is critical for apoptosis.There are two canonical apoptotic pathways, differing in that theapoptosis-initiating stimulus is intracellular (intrinsic pathway) orextracellular (extrinsic pathway). These pathways converge at thecleavage of procaspase-3 to form the active caspase-3, the key“executioner” caspase that catalyzes the hydrolysis of hundreds ofprotein substrates, leading to cell death.

One of the hallmarks of cancer is the ability of cancer cells to evadeapoptosis, allowing for unchecked proliferation. As such, reactivationof apoptosis in cells with defective apoptotic pathways is a promisinganticancer strategy. Compounds such as p53-MDM2 disruptors (Nutlins),Bcl-2 inhibitors (ABT-737), and inhibitors of XIAP (SM-164) all actdirectly on proteins in the apoptotic cascade, inducing apoptosis andleading to death of cancer cells.

Complementary to the strategies described above, the direct activationof procaspase-3 with a small molecule has potential for personalizingcancer therapy. Procaspase-3 levels are elevated in certain cancers,including lymphomas, leukemias, melanomas, pancreatic cancer, livercancers, lung cancers, breast cancers, and colon cancers. Due to theelevated levels of procaspase-3 in cancer cells, the requirement ofcaspase-3 activation for apoptosis, and the relative downstream locationof procaspase-3 in the apoptotic cascade, induction of apoptosis by thedirect activation of procaspase-3 is being actively explored as apersonalized anticancer strategy. Accordingly, there is a need for newcompounds that modulate procaspase-3 activity, particularly compoundsthat activate procaspase-3 and that are metabolically stable enough tobe effective clinical therapies.

SUMMARY

Procaspase-Activating Compound 1 (PAC-1) is an ortho-hydroxy N-acylhydrazone that enhances the enzymatic activity of procaspase-3 in vitroand induces apoptosis in cancer cells. An analogue of PAC-1, calledS-PAC-1, was evaluated in a veterinary clinical trial in pet dogs withlymphoma and found to have considerable potential as an anticanceragent. With the goal of identifying more potent compounds in thispromising class of experimental therapeutics, a combinatorial librarybased on PAC-1 was created, and the compounds were evaluated for theirability to induce death of cancer cells in culture. The compounds wereevaluated for their ability to induce apoptosis in cancer cells and fortheir metabolic stability. The newly identified compounds can providetherapeutics for treatment of the many cancers that have elevatedexpression levels of procaspase-3.

Compounds capable of activating enzymes that are often overexpressed intheir inactive form in cancer cells have been discovered. The compoundscan induce programmed cell death (apoptosis) in cancer cells, includingthose that have upregulated procaspase-3. Many cancers resist standardchemotherapy. The compounds described herein can take advantage ofbiological targets that may be upregulated in cancer cells and thus canbe effective even in cells with defects in their apoptotic machinery.These compounds can also be successful in targeted cancer therapy byselectively killing cancer cells with comparably reduced adversereactions to non-cancerous cells having lower levels of procaspase-3.These adverse reactions can include toxicity, particularlyneurotoxicity.

Accordingly, the invention provides compounds of compound of Formula I:

wherein

R¹ is an optionally substituted benzoyl, phenyl, (aryl)methylene, or(aryl)methine wherein the methine carbon is optionally substituted withphenyl;

n is 1, 2, 3, or 4; and

each R² is independently H, alkyl, alkoxy, hydroxy, carboxy, halo,amino, alkylamino, dialkylamino, trifluoromethyl, trifluoromethoxy,benzyl, benzyloxy, nitro, cyano (—CN), sulfonamide (—SO₂NH₂),2-propenyl, acetylene, N-alkyl-triazole, or N-benzyl-triazole; or two R²groups form an ortho-fused benzo group;

or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, R¹ is benzoyl (Ph(C═O)—). In other embodiments, R¹is a substituted benzoyl. The benzoyl group can be substituted with 1,2, 3, or 4 R² groups. The variables R² can be ortho, meta, or para, or acombination thereof, to the carbonyl of the benzoyl group.

In some embodiments, n is 1 or 2. In other embodiments, n is 3 or 4. Thevariables R² can be ortho, meta, or para to the hydroxyl group ofFormula I, or a combination thereof.

In some embodiments, R² is methyl, t-butyl, methoxy, hydroxy, fluoro,chloro, bromo, iodo, amino, ethylamino, diethylamino, trifluoromethyl,trifluoromethoxy, benzyl, benzyloxy, nitro, cyano, sulfonamide,2-propenyl, acetylene, N-methyl-triazole, or N-benzyl-triazole. Invarious embodiments, n is 2 and two R² groups form an ortho-fused benzogroup. In some embodiments, a substituent on an R¹ phenyl group can be asubstituent R². In various embodiments, R² can independently be asubstituent on an R¹ aryl group, including a benzoyl group, and suchgroups can have one to five R² substituents.

In some embodiments, n is 2 and each R² is t-butyl.

In some embodiments, n is 1 and R² is 2-propenyl.

In some embodiments, R¹ is a methoxy-benzyl; dimethoxy-benzyl;benzyloxy-benzyl; t-butyl-benzyl; naphthylmethylene; or ethyl-benzyl.

In certain specific embodiments, R¹ is 4-methoxy-benzyl;2,5-dimethoxy-benzyl; 4-benzyloxy-benzyl; 4-t-butyl-benzyl;2-naphthylmethylene; or 4-ethyl-benzyl.

In certain other specific embodiments, R¹ is:

In various specific embodiments, the compound is one or more ofcompounds 1-45 of Example 4, a compound of Table 1, or apharmaceutically acceptable salt or solvate thereof. In otherembodiments, the compound is a compound described herein wherein themethylene carbon attached to the distal piperazine nitrogen issubstituted with an oxo group, for example:

and the like, for each compound described or illustrated herein withoutan oxo group at the methylene carbon attached to the distal piperazinenitrogen. For example, in one embodiment, the compound is a compound ofFormula (X):

wherein

R¹⁰ is H, F, Cl, Br, —NO₂, —CN, —CF₃, —OCF₃, or —SO₂NH₂;

R²⁰ is H, F, Cl, Br, —NO₂, —CN, —CF₃, —OCF₃, or —SO₂NH₂; and

R³⁰ is H, (C₁-C₆)alkyl, (C₁-C₆)alkenyl, or (C₁-C₆)alkoxy;

or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, R¹⁰ is H.

In some embodiments, R¹⁰ is F, Cl, or Br.

In some embodiments, R¹⁰ is —NO₂ or —CN.

In some embodiments, R¹⁰ is —CF₃, —OCF₃, or —SO₂NH₂.

In some embodiments, R²⁰ is H. In some embodiments, R²⁰ is F. In someembodiments, R²⁰ is H or F.

In some embodiments, R²⁰ is F, Cl, or Br.

In some embodiments, R²⁰ is —NO₂ or —CN.

In some embodiments, R²⁰ is —CF₃, —OCF₃, or —SO₂NH₂.

In some embodiments, R³⁰ is H.

In some embodiments, R³⁰ is n-propyl.

In some embodiments, R³⁰ is 2-propenyl(allyl).

In some embodiments, R¹⁰ can be R¹ as described above, and vice versa.

In some embodiments, R²⁰ can be R² as described above, and vice versa.

In some embodiments, R³⁰ can be R³ as described above, and vice versa.

The invention also provides a pharmaceutical composition comprising acompound described herein and a pharmaceutically acceptable diluent,excipient, or carrier. In some embodiments, the compound induces deathof cancer cells in culture.

The invention further provides a method of treating a cancer cellcomprising (a) identifying a susceptibility to treatment of a cancercell with a procaspase activator compound; and (b) exposing a cancercell to an effective amount of the procaspase activator compound;wherein the procaspase activator compound is a compound describedherein.

The invention additionally provides a method of inducing apoptosis in acell comprising administering to a cell an effective amount of acompound described herein.

In some embodiments, the invention provides compounds and methodsinvolving effective concentrations, such as about 10 nM to about 100 μMof the compound or formula. In some embodiments, the effectiveconcentrations are from about 200 nM to about 5 μM. In anotherembodiment, the effective concentration is a value such as a 50%activity concentration in a direct procaspase activation assay, in acell apoptosis induction assay, or in an animal clinical therapeuticassessment. In another embodiment, such value is less than about 200 μM.In various embodiments, the value is less than about 10 μM. In variousembodiments, a compound can have significant metabolic stability. Forexample, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 90%, or at least about 95% of a sample of thecompound can remain after a 3-hour incubation in a liver microsomestability assay.

The invention therefore provides compounds, compositions, and methods oftherapeutic treatment. In some embodiments, the inventions areapplicable in the context of a variety of cancer diseases and cancercell types such as breast, lymphoma, adrenal, renal, melanoma, leukemia,neuroblastoma, lung, brain, among others.

The invention provides the novel compounds described herein and thecompounds of the Formulas described herein, intermediates for thesynthesis of such compounds, as well as methods of preparing thecompounds. The invention also provides compounds that are useful asintermediates for the synthesis of other useful compounds.

The also invention provides for the use of the compositions describedherein for use in medical therapy. The medical therapy can be treatingcancer, for example, lymphomas, leukemias, melanomas, pancreatic cancer,liver cancers, lung cancers, breast cancers, and colon cancers. Theinvention also provides for the use of a composition as described hereinfor the manufacture of a medicament to treat a disease in a mammal,e.g., cancer in a human. The medicament can include a pharmaceuticallyacceptable diluent, excipient, or carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention. However, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1. Hydrazides used to construct a combinatorial library of PAC-1analogues. Each R¹ benzyl hydrazide precursor can also be acorresponding R¹ benzoyl hydrazide precursor compound, in variousembodiments.

FIG. 2. Aldehydes used to a combinatorial library of PAC-1 analogues,according to certain embodiments.

FIG. 3. Cell culture activity. U-937 cells (human lymphoma) treated withcompounds at 25 μM for 24 hours. Cell viability assessed by Alamar blueassay. Error bars represent standard error from the mean (n=3).

FIG. 4. Liver Microsome Stability Assay. Compounds (10 μM) incubatedwith liver microsomes for 3 h and quenched with MeCN containing internalstandard, analyzed by LC/MS (280 nm).

DETAILED DESCRIPTION

In 2006, the discovery of Procaspase-Activating Compound 1 (PAC-1,Scheme A) was reported (Putt et al., Nat Chem Biol 2006, 2, 543-550).PAC-1 enhances the enzymatic activity of procaspase-3 in vitro, inducesapoptotic cell death in cancer cells, and shows efficacy in multiplemurine tumor models. Structure-activity relationship studies revealedthat the activity of PAC-1 in vitro and in cell culture is dependent onthe presence of the ortho-hydroxy N-acyl hydrazone moiety (Scheme A), afunctional group known to participate in metal chelation. Indeed, zincis a powerful inhibitor of procaspase-3 enzymatic activity, and themechanism by which PAC-1 activates procaspase-3 in vitro is throughchelation of inhibitory zinc from procaspase-3, which allowsprocaspase-3 to process itself to the active form. This same basicmechanism appears to be operational in cell culture as well:approximately 10% of cellular zinc is not bound tightly but exists asthe “labile zinc pool”. As zinc from the labile pool has been shown toco-localize with procaspase-3, it appears that PAC-1 chelation of thislabile zinc inside the cells enhances procaspase-3 activity, leading toapoptosis.

PAC-1 can be safely administered to mice and research dogs at doses thatgive serum concentrations of ˜10 μM for 48 hours. Asulfonamide-containing derivative of PAC-1, called S-PAC-1 (Scheme A),can be safely administered at doses that provide very high serumconcentrations in mice (˜3.5 mM). Encouragingly, a veterinary clinicaltrial of S-PAC-1 (administered as a 24- or 72-hour continuous IVinfusion) in pet dogs with spontaneously-occurring lymphoma revealedthis compound to be safe in all veterinary patients and effective atreducing or stabilizing tumor growth in 4 out of 6 patients. This resultprovides proof-of-concept for the notion that procaspase-3 activationvia small molecule chelation of labile zinc can be a safe and effectiveanticancer strategy. In the continued search for more potent derivativesof PAC-1, we report herein the synthesis of new PAC-1 analogues, theevaluation of these compounds for their ability to induce death ofcancer cells in culture, and further characterization various compoundswith heightened metabolic stability.

Design and Synthesis of Combinatorial Library Based on PAC-1.

A library of PAC-1 analogues was designed with the goal of identifyingcompounds capable of eliciting potent death of cancer cells in culture.As the maximal cytotoxicity of S-PAC-1 is not reached until at least 24hours, and both PAC-1 and S-PAC-1 exhibit short half-lives of 1-2 hoursin vivo, a secondary goal of this study was to identify PAC-1 analoguesthat could induce apoptosis more rapidly. Reported synthetic routes toPAC-1 and S-PAC-1, as well as other PAC-1 analogues, utilize thecondensation of a hydrazide and an aldehyde as the final step in thesynthetic scheme (U.S. Patent Publication No. 2007/0049602) (WO2008/134474 (Hergenrother et al.)). This modular nature of the PAC-1synthesis allows for a diverse array of functional groups to beconveniently incorporated into the PAC-1 scaffold without altering thecore ortho-hydroxy N-acyl hydrazone motif essential for procaspase-3activation and induction of apoptosis.

As shown in FIGS. 1 and 2, 31 hydrazides (1{1-31}) and 27 aldehydes(2{1-27}) were selected for building the library of 837 PAC-1 analogues.The hydrazides were constructed from commercially available benzylhalide starting materials. The syntheses of hydrazides 1{1-6} have beenreported previously (Putt et al., Nat Chem Biol 2006, 2, 543-550;Peterson et al., J Med Chem 2009, 52, 5721-5731; Peterson et al., CancerRes 2010, 70, 7232-41). Hydrazides 1{7-27} were synthesized according toScheme 1. Substituted benzyl halides 4 {7-27} first reacted withpiperazine to form substituted benzylpiperazines 5{7-27}. A secondalkylation of the piperazine ring with ethyl chloroacetate gavedisubstituted piperazines 6 {7-27}, and the esters were then convertedto hydrazides 1{7-27} by reaction with hydrazine.

The synthetic routes toward hydrazides 1{28-31} are detailed in Scheme2. Synthesis of hydrazide 1{28} began by the alkylation of piperazinewith 4-vinylbenzyl chloride (7) to form monosubstituted piperazine 8(Scheme 2, equation 1). A second alkylation with ethyl chloroacetateformed ester 9, and reaction with hydrazine formed the hydrazide andreduced the olefin, giving hydrazide 1{28}. The reduction of olefinswith hydrazine typically involves the addition of an oxidizing agent(Miller, C. E., Hydrogenation with Diimide. J Chem Educ 1965, 42, 254),but the presence of atmospheric oxygen was sufficient to achieve thistransformation.

Synthesis of hydrazide 1{29} (Scheme 2, equation 2) began with thereaction of ethyl 2-(piperazin-1-yl)acetate (10) with benzyl bromide 4{29} to form intermediate 6 {29}. Reaction of 6 {29} with hydrazine thenformed hydrazide 1{29}. Hydrazide 1 {30} (Scheme 2, equation 3) wassynthesized beginning with the reaction of 1-phenylpiperazine (5{30})with ethyl chloroacetate to give disubstituted piperazine 6{30}, andreaction with hydrazine formed hydrazide 1{30}. Hydrazide 1{31}, wassynthesized by first protecting 4-methylbenzophenone (11) as theethylene acetal (12), as shown in Scheme 2, equation 4. This compoundwas brominated under radical conditions to give benzyl bromide 13.Reaction with monosubstituted piperazine 10 gave intermediate 14, andreaction with hydrazine gave hydrazide 15. Deprotection of the acetalwith aqueous acid gave hydrazide 1{31}.

The structure-activity relationship of PAC-1 derived from the synthesisand evaluation of 30 compounds demonstrated the necessity of theortho-hydroxyl group, so 27 salicylaldehyde building blocks wereselected for library construction. Aldehydes 2{1-23} were obtained fromcommercial sources, and the syntheses of aldehydes 2{24-26} have beenreported previously (Peterson et al., J Med Chem 2009, 52, 5721-5731;Peterson et al., Cancer Res 2010, 70, 7232-41; Chang et al., DaltonTrans 2004, 1731-8). Aldehyde 2{27} was synthesized via copper-catalyzedcycloaddition of aldehyde 2{26} with benzyl azide, as shown in Scheme 3.

Using a Büchi Syncore parallel synthesizer, each hydrazide was condensedwith each aldehyde, with over 80 reactions performed simultaneously.Each aldehyde (5-15 mg) was allowed to react with excess hydrazide (1.7equiv), and mass spectrometry was used to monitor the disappearance ofthe aldehyde from the reaction mixture. When the aldehyde had reactedcompletely, polystyrene-bound benzaldehyde was added as a scavengerresin to react with and remove the excess hydrazide. When massspectrometry showed no hydrazide remaining, the beads were filtered, andthe solutions were dried under high vacuum. Each of the 837 compoundswas assessed by HPLC/MS. The purity of each compound was about 74-100%,with an average purity of 91%.

Evaluation of the PAC-1 Combinatorial Library.

With 837 PAC-1 analogues in hand, compounds were evaluated for theirability to induce apoptosis in cell culture. U-937 human lymphoma cellswere exposed to the compounds for 24 hours at a concentration of 20 μM.Both PAC-1 and S-PAC-1 display moderate potency (˜50% cell death) versusthis cell line under these conditions. Apoptotic cell death was assessedby flow cytometry, using Annexin V-FITC/propidium iodide staining.Through this screening process, six compounds were identified andconfirmed to induce >80% cell death under these conditions.

TABLE A Six library compounds induce potent cell death of U-937 cells(human lymphoma) in both 24 and 72 hour experiments, with biomassquantified using the sulforhodamine B assay. 72-hour % CytotoxicityProcaspase-3 IC₅₀ (μM) (24 hours at 7.5 μM) (% Activity at 3.5 μM)

3.8 ± 0.4 21 42 ± 1.8 PAC-1

4.4 ± 0.7 23 4 ± 0.6 S-PAC-1

1.8 ± 0.2 90 53 ± 4.1 3{2,7}

1.6 ± 0.2 53 64 ± 2.5 3{4,7}

1.4 ± 0.2 97 36 ± 1.6 3{18,7}

0.9 ± 0.03 83 82 ± 2.4 3{20,24}

1.0 ± 0.04 50 69 ± 5.3 3{25,7}

2.0 ± 0.2 70 60 ± 2.4 3{28,7}

Cell Death Induction and Relief of Zinc-Mediated Inhibition ofProcaspase-3 by Hit Compounds.

After re-synthesis of the hits (3 {2,7}, 3 {4,7}, 3{18,7}, 3 {20,24}, 3{25,7}, and 3{28,7}), analytically pure samples of the compounds wereevaluated in further biological assays. These structures and thebiological results are shown in Table A, above. The compounds wereevaluated, at a range of concentrations, for their ability to inducecell death in U-937 cells, as well as their ability to activateprocaspase-3 in vitro. All six of these hits were found to be 2-4 foldmore potent in cell culture than PAC-1 and S-PAC-1 in a 72-hourtreatment.

In a second experiment, flow cytometry analysis with AnnexinV-FITC/propidium iodide was performed on U-937 cells that were exposedto the compounds at a single concentration (7.5 μM) for 24 hours (TableA). Within 24 hours the majority of the compound treated cells wereundergoing apoptosis (cells in the lower right quadrant of thehistogram—Annexin V positive, propidium iodide negative), or were in alate apoptotic/necrotic stage (upper right quadrant—Annexin V positive,propidium iodide positive). The novel analogues were found to be morepotent than PAC-1 under these 24 hour conditions.

The six confirmed hits were then evaluated in vitro for their ability torelieve zinc-mediated inhibition of procaspase-3 (Table A). In thisexperiment, procaspase-3 was incubated with ZnSO₄, conditions in whichprocaspase-3 has no enzymatic activity. All compounds were able toenhance procaspase-3 enzymatic activity under these conditions (asassessed by the cleavage of the colorimetric caspase-3 substrateAc-DEVD-pNA, synthesized as previously reported (Peterson et al., NatProtoc 2010, 5, 294-302)), and five of the six hit compounds showedgreater activity than PAC-1 in this assay. These data indicate that thecompounds enhance the activity of procaspase-3 in vitro throughchelation of inhibitory zinc, and suggest that in the cell the compoundschelate zinc from the labile pool, allowing procaspase-3 to be processedto active caspase-3, leading to apoptotic cell death.

The direct modulation of apoptotic proteins is a practical anticancerstrategy. PAC-1 and its derivative S-PAC-1, which chelate labilecellular zinc and induce apoptosis in cancer cells, have been effectivein various preclinical anti-tumor models. However, derivatives thatinduce cell death more rapidly and more potently would be even moreattractive as therapeutics. Using parallel synthesis and guided by theknown SAR, we constructed 837 PAC-1 analogues and evaluated them fortheir cell death inducing properties. The six compounds shown in Table Aemerged from this effort. These compounds are two- to four-fold morepotent than PAC-1 at induction of cancer cell death in both 24-hour and72-hour assays.

Given the general hydrophobicity of the hit compounds relative to PAC-1,the enhanced potency and enhanced rate of cell death may be driven byenhanced cell permeability. These qualities are likely to beadvantageous as the compounds are moved forward in vivo. In addition,other members of this library will likely emerge as viable in vivocandidates as alternate properties (such as propensity to cross theblood-brain barrier, improved metabolic stability, improvedsolubility/formulation for in vivo studies, etc.) are examined. Thus,this library of 837 compounds will be a rich source from which todevelop next-generation procaspase-3 activating compounds.

Additional Compounds and Analysis.

A high-throughput screen of approximately 20,000 compounds identifiedPAC-1 (1, Scheme A1) as a compound that enhanced the cleavage ofprocaspase-3 in vitro. The compound induces apoptotic cell death in awide array of cancer cell lines in culture and shows anticancer efficacyin multiple murine tumor models.¹ Further study of thestructure-activity relationships (SAR) identified theortho-hydroxy-N-acylhydrazone as the key pharmacophore.²⁻³ Several PAC-1derivatives containing this motif have comparable activity in vitro andin cell culture, but derivatives with a modified core lose activity.³The ortho-hydroxy-N-acylhydrazone is known to chelate metals,⁴ many ofwhich are also known to inhibit procaspase and caspase enzymes.^(2, 5-7)In particular, zinc from the labile zinc pool, which is bound looselyand does not play an essential role in the activity of these proteins,has been shown to colocalize with procaspase-3 and inhibit its enzymaticactivity. The mechanism of action of PAC-1 likely involves the chelationof zinc from the labile pool, relieving the zinc-mediated inhibition ofprocaspase-3 and allowing the enzyme to process itself to the activeform.²⁻³

Pharmacokinetic studies with PAC-1 revealed that serum concentrations ofapproximately 10 μM can be achieved with minimal side effects.⁸ Asulfonamide-containing derivative of PAC-1, called S-PAC-1 (2, SchemeA1), can be safely administered at doses of 350 mg/kg or higher, givinga peak plasma concentration of 3.5 mM.⁹ The improved safety profile isdue in large part to its decreased ability to cross the blood-brainbarrier (BBB), as compared to PAC-1.¹⁰ Encouragingly, S-PAC-1 waseffective in reducing or stabilizing tumor growth in four out of sixcanine patients with spontaneously occurring lymphoma, and the compoundwas well tolerated in all six dogs.⁹ This result demonstrates thepotential for procaspase activation as a safe and effective anticancerstrategy.

Results and Discussion.

Compound Synthesis.

Previous syntheses of PAC-1 and other derivatives involved thelate-stage condensation of a hydrazide and an aldehyde to form the keyortho-hydroxy-N-acylhydrazone.^(1,3,9,11) This reaction has been usefulfor the generation of large numbers of derivatives from a comparativelysmall number of starting materials.¹¹ In this work, PAC-1 analogues 1-45were synthesized by the condensation of nine hydrazides (46a-i) withfive aldehydes (47a-e), as shown in Scheme A2.

The hydrazides were synthesized according to Scheme A3a-c. The synthesisbegan with the alkylation of piperazine (48) with ethyl chloroacetate(49) to form monosubstituted piperazine 50. Compound 50 was then reactedwith a substituted benzyl or benzoyl halide to give disubstitutedpiperazines 51a-i in high yields. Reaction of the esters with hydrazinethen gave hydrazides 46a-i.

Synthesis of the aldehydes is shown in Scheme A3b. Both salicylaldehyde(52) and 5-fluorosalicylaldehyde (47c) were alkylated with allyl bromideto give allyloxybenzaldehydes 53a-b in high yields. Heating thesecompounds at 200° C. allowed for these substrates to undergo Claisenrearrangements to give aldehydes 47a and 47d in approximately 50% yield.Finally, chemoselective hydrogenation with diphenyl sulfide as acatalyst poison¹² gave aldehydes 47b and 47e in high yield. As shown inScheme A3c, each of the hydrazides (46a-i) was condensed with each ofthe aldehydes (47a-e) in the presence of catalytic HCl to give PAC-1derivatives 1-45, the structures of which are given in Table 1 (see alsoFIGS. 3 and 4).

TABLE 1 Structures, experimental data, and predicted logBB values ofPAC-1 analogues. 1-45

U-937 RLM Predicted Mouse compound R¹ R² R³ 72 h IC₅₀ (μM) 3 h %Stability logBB Toxicity  1 (PAC-1) Bn H All 10.2 ± 0.3 38 ± 2 −0.36 3 2 (S-PAC-1) 4-SO₂NH₂—Bn H All  8.9 ± 0.6 84 ± 0 −1.45 0  3 Bz H All12.1 ± 1.3 89 ± 4 −0.72 3  4 4-CN—Bn H All 13.7 ± 0.9 48 ± 2 −0.73 1  54-CN—Bz H All 13.1 ± 3.7 90 ± 4 −1.09 death >72 h  6 4-F—Bn H All 11.1 ±2.1 31 ± 1 −0.33 3  7 4-F—Bz H All 10.2 ± 1.7 86 ± 2 −0.69 2  8 4-CF₃—BnH All 15.3 ± 6.7 16 ± 1 −0.22 death <24 h  9 4-CF₃—Bz H All  6.6 ± 1.985 ± 6 −0.57 death <24 h* 10 Bn H n-Pr  9.6 ± 2.1 30 ± 1 −0.30 2 114-SO₂NH₂—Bn H n-Pr  4.9 ± 0.4 61 ± 2 −1.39 — 12 Bz H n-Pr  9.4 ± 1.3 71± 3 −0.66 — 13 4-CN—Bn H n-Pr  9.0 ± 1.2 30 ± 2 −0.67 — 14 4-CN—Bz Hn-Pr 12.8 ± 2.7 61 ± 3 −1.03 — 15 4-F—Bn H n-Pr 10.0 ± 1.7 24 ± 2 −0.27death <24 h 16 4-F—Bz H n-Pr  7.3 ± 0.9 69 ± 4 −0.63 — 17 4-CF₃—Bn Hn-Pr  4.1 ± 0.4 15 ± 2 −0.16 death >72 h 18 4-CF₃—Bz H n-Pr  4.8 ± 1.264 ± 1 −0.51 death <24 h* 19 Bn F H 17.0 ± 1.4 64 ± 4 −0.49 3 204-SO₂NH₂—Bn F H 19.6 ± 3.8 85 ± 6 −1.57 death >72 h 21 Bz F H 15.7 ± 2.688 ± 1 −0.84 — 22 4-CN—Bn F H 13.5 ± 1.0 79 ± 4 −0.86 death <24 h 234-CN—Bz F H 15.3 ± 1.3 88 ± 4 −1.21 — 24 4-F—Bn F H 11.7 ± 1.4 62 ± 2−0.45 3 25 4-F—Bz F H 15.3 ± 0.8 86 ± 2 −0.81 — 26 4-CF₃—Bn F H  4.7 ±0.3 30 ± 5 −0.34 3 27 4-CF₃—Bz F H  8.7 ± 0.5 87 ± 3 −0.70 2 28 Bn F All 9.5 ± 0.9 56 ± 1 −0.33 death <24 h 29 4-SO₂NH₂—Bn F All  9.8 ± 1.3 89 ±3 −1.42 death >72 h 30 Bz F All  8.6 ± 2.0 93 ± 7 −0.69 2 31 4-CN—Bn FAll 12.7 ± 2.0 65 ± 2 −0.70 — 32 4-CN—Bz F All 10.1 ± 2.0 95 ± 4 −1.06 133 4-F—Bn F All 10.3 ± 4.1 57 ± 1 −0.30 — 34 4-F—Bz F All  8.5 ± 1.4 92± 3 −0.65 3 35 4-CF₃—Bn F All  3.4 ± 0.6 49 ± 3 −0.19 death <24 h* 364-CF₃—Bz F All  6.5 ± 0.6 90 ± 2 −0.54 2 37 Bn F n-Pr  8.9 ± 1.2 49 ± 6−0.27 death <24 h 38 4-SO₂NH₂—Bn F n-Pr  8.7 ± 0.4 62 ± 3 −1.36 — 39 BzF n-Pr 12.3 ± 1.0 86 ± 5 −0.63 2 40 4-CN—Bn F n-Pr 11.2 ± 0.9 49 ± 5−0.64 — 41 4-CN—Bz F n-Pr  9.4 ± 1.2 66 ± 3 −1.00 1 42 4-F—Bn F n-Pr 7.5 ± 0.7 48 ± 1 −0.24 — 43 4-F—Bz F n-Pr  7.5 ± 1.4 67 ± 3 −0.59 3 444-CF₃—Bn F n-Pr  3.9 ± 0.6 40 ± 1 −0.13 death <24 h 45 4-CF₃—Bz F n-Pr 5.2 ± 0.6 64 ± 5 −0.48 death >72 h

Evaluation of PAC-1 Analogues.

After the synthesis was complete, the biological activity of thecompounds was evaluated. First, the 72-hour IC₅₀ values of the compoundsagainst U-937 cells in culture were determined (Table 1). Encouragingly,each of the compounds was found to induce dose-dependent cell deathunder these conditions, and most of the compounds were approximately aspotent as PAC-1 and S-PAC-1.

Next, the metabolic stability of the compounds was evaluated in ratliver microsomes. The compounds were evaluated for 3 hours at 10 μM, andthe metabolites were observed by LC/MS. The results of this assay areshown in Table 1. Compounds that contained benzoyl substituents weresignificantly more stable than analogous compounds containing benzylgroups. Unexpectedly, the propyl-containing compounds were less stablethan the allyl-containing compounds, although the dihydroxylatedmetabolites were not observed with the propyl compounds. In addition,S-PAC-1 was reasonably stable in the liver microsomes, despite the shortin vivo half-life of the compound, suggesting that other clearancemechanisms play a greater role in elimination of S-PAC-1 from the body.

Finally, the predicted log BB value was calculated for each compound.This algorithm, which involves polar surface area and ClogP, is used topredict the permeability of small molecules across the blood-brainbarrier (BBB).¹³ Compounds with more positive log BB values will havehigher concentrations in the brain, while compounds with more negativelog BB values will have higher concentrations in the blood. The valuesare shown in Table 1. As expected, the compounds containing morehydrophobic substituents are predicted to cross the BBB to a greaterdegree than those containing more polar substituents.

Assessment of Toxicity In Vivo.

With the goal of identifying a compound with improved tolerability, 32of the 45 PAC-1 analogues were evaluated in mice. The results of thetoxicity study are shown in Table 1. The level of toxicity was rated ona scale of 0 (no observable adverse effect) to 3 (severe toxicity,almost fatal). Many of the compounds were lethal to the mice, as themice died either within 24 hours of treatment or greater than 72 hourspost-treatment; these results are also noted.

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DEFINITIONS

As used herein, the recited terms have the following meanings. All otherterms and phrases used in this specification have their ordinarymeanings as one of skill in the art would understand. Such ordinarymeanings may be obtained by reference to technical dictionaries, such asHawley's Condensed Chemical Dictionary 14^(th) Edition, by R. J. Lewis,John Wiley & Sons, New York, N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,etc., indicate that the embodiment described may include a particularaspect, feature, structure, moiety, or characteristic, but not everyembodiment necessarily includes that aspect, feature, structure, moiety,or characteristic. Moreover, such phrases may, but do not necessarily,refer to the same embodiment referred to in other portions of thespecification. Further, when a particular aspect, feature, structure,moiety, or characteristic is described in connection with an embodiment,it is within the knowledge of one skilled in the art to affect orconnect such aspect, feature, structure, moiety, or characteristic withother embodiments, whether or not explicitly described.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds, so that acompound X includes a plurality of compounds X. It is further noted thatthe claims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely,” “only,” and the like, inconnection with the recitation of claim elements or use of a “negative”limitation.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrase “one or more” is readily understood by one of skill in the art,particularly when read in context of its usage. For example, one or moresubstituents on a phenyl ring refers to one to five, or one to four, forexample if the phenyl ring is disubstituted.

The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer at each end of the range. Unless indicatedotherwise herein, the term “about” is intended to include values, e.g.,weight percents, proximate to the recited range that are equivalent interms of the functionality of the individual ingredient, thecomposition, or the embodiment.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges recited herein also encompass any and all possible sub-ranges andcombinations of sub-ranges thereof, as well as the individual valuesmaking up the range, particularly integer values. A recited range (e.g.,weight percents or carbon groups) includes each specific value, integer,decimal, or identity within the range. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths, ortenths. As a non-limiting example, each range discussed herein can bereadily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art, all languagesuch as “up to”, “at least”, “greater than”, “less than”, “more than”,“or more”, and the like, include the number recited and such terms referto ranges that can be subsequently broken down into sub-ranges asdiscussed above. In the same manner, all ratios recited herein alsoinclude all sub-ratios falling within the broader ratio. Accordingly,specific values recited for radicals, substituents, and ranges, are forillustration only; they do not exclude other defined values or othervalues within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, theinvention encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group. Additionally, for all purposes, the invention encompassesnot only the main group, but also the main group absent one or more ofthe group members. The invention therefore envisages the explicitexclusion of any one or more of members of a recited group. Accordingly,provisos may apply to any of the disclosed categories or embodimentswhereby any one or more of the recited elements, species, orembodiments, may be excluded from such categories or embodiments, forexample, as used in an explicit negative limitation.

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity, including at the cellularor molecular level, for example, to bring about a physiologicalreaction, a chemical reaction, or a physical change, e.g., in asolution, in a reaction mixture, in vitro, or in vivo.

An “effective amount” refers to an amount effective to treat a disease,disorder, and/or condition, or to bring about a recited effect, such asactivation or inhibition. For example, an effective amount can be anamount effective to reduce the progression or severity of the conditionor symptoms being treated. Determination of a therapeutically effectiveamount is well within the capacity of persons skilled in the art. Theterm “effective amount” is intended to include an amount of a compounddescribed herein, or an amount of a combination of compounds describedherein, e.g., that is effective to treat or prevent a disease ordisorder, or to treat the symptoms of the disease or disorder, in ahost. Thus, an “effective amount” generally means an amount thatprovides the desired effect. In one embodiment, an effective amountrefers to an amount of the active agent described herein that areeffective, either alone or in combination with a pharmaceutical carrier,upon single- or multiple-dose administration to a cell or a subject,e.g., a patient, at inhibiting the growth or proliferation, inducing thekilling, or preventing the growth of hyperproliferative cells. Suchgrowth inhibition or killing can be reflected as a prolongation of thesurvival of the subject, e.g., a patient beyond that expected in theabsence of such treatment, or any improvement in the prognosis of thesubject relative to the absence of such treatment.

The terms “treating”, “treat” and “treatment” include (i) preventing adisease, pathologic or medical condition from occurring (e.g.,prophylaxis); (ii) inhibiting the disease, pathologic or medicalcondition or arresting its development; (iii) relieving the disease,pathologic or medical condition; and/or (iv) diminishing symptomsassociated with the disease, pathologic or medical condition. Thus, theterms “treat”, “treatment”, and “treating” can extend to prophylaxis andcan include prevent, prevention, preventing, lowering, stopping orreversing the progression or severity of the condition or symptoms beingtreated. As such, the term “treatment” can include medical, therapeutic,and/or prophylactic administration, as appropriate. In some embodiments,the terms “treatment”, “treat” or “treated” can refer to (i) preventionof tumor growth or regrowth of the tumor (prophylaxis), (ii) a reductionor elimination of symptoms or the disease of interest (therapy) or (iii)the elimination or destruction of the tumor (cure).

The terms “inhibit”, “inhibiting”, and “inhibition” refer to theslowing, halting, or reversing the growth or progression of a disease,infection, condition, or group of cells. The inhibition can be greaterthan about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, comparedto the growth or progression that occurs in the absence of the treatmentor contacting. Additionally, the terms “induce,” “inhibit,”“potentiate,” “elevate,” “increase,” “decrease,” or the like denotequantitative differences between two states, and can refer to at leaststatistically significant differences between the two states. Forexample, “an amount effective to inhibit the growth ofhyperproliferative cells” means that the rate of growth of the cells canbe, in some embodiments, at least statistically significantly differentfrom the untreated cells. Such terms can be applied herein to, forexample, rates of proliferation.

The phrase “inhibiting the growth or proliferation” of thehyperproliferative cell, e.g. neoplastic cell, refers to the slowing,interrupting, arresting, or stopping its growth and metastasis, and doesnot necessarily indicate a total elimination of the neoplastic growth.

The term “cancer cell” encompasses definitions as broadly understood inthe art. In one embodiment, the term refers to an abnormally regulatedcell that can contribute to a clinical condition of cancer in a human oranimal. In some embodiments, the term can refer to a cultured cell lineor a cell within or derived from a human or animal body. A cancer cellcan be of a wide variety of differentiated cell, tissue, or organ typesas is understood in the art, and also as described herein.

Thus, the term “cancer” generally refers to any of a group of more than100 diseases caused by the uncontrolled growth of abnormal cells. Cancercan take the form of solid tumors and lymphomas, and non-solid cancerssuch as leukemia. Unlike normal cells, which reproduce until maturationand then only as necessary to replace wounded cells, cancer cells cangrow and divide endlessly, crowding out nearby cells and eventuallyspreading to other parts of the body.

The invention provides methods for treating cancer and cancerousconditions. The term “cancerous condition” relates to any conditionwhere cells are in an abnormal state or condition that is characterizedby rapid proliferation or neoplasia. A cancerous condition may bemalignant or non-malignant (e.g. precancerous condition) in nature. Tofarther describe a “cancerous condition”, the terms“hyperproliferative”, “hyperplastic”, “hyperplasia”, “malignant”,“neoplastic” and “neoplasia” can be used. These terms can be usedinterchangeably and are meant to include all types of hyperproliferativegrowth, hyperplastic growth, cancerous growths or oncogenic processes,metastatic tissues or malignantly transformed cells, tissues or organs,irrespective of histopathologic type, stage of invasiveness, orcancerous determination (e.g. malignant and nonmalignant).

The term “neoplasia” refers to new cell growth that results in a loss ofresponsiveness to normal growth controls, e.g., neoplastic cell growth.A “hyperplasia” refers to cells undergoing an abnormally high rate ofgrowth. However, these terms can be used interchangeably, as theircontext will reveal, referring generally to cells experiencing abnormalcell growth rates. “Neoplasias” and “hyperplasias” include tumors, whichmay be either benign, premalignant, carcinoma in-situ, malignant, solidor non-solid.

Compounds described herein have been found to be particularly effectivefor treating cancers of the brain. Cancers of the brain include, but arenot limited to, oligodendrogliomas and glioblastomas includingglioblastoma multiforme (GBM). Tissues affected by the cancerous cellscan be in the brain itself (e.g., the cranium or the central spinalcanal) or in lymphatic tissue, in blood vessels, in the cranial nerves,in the brain envelopes (meninges), skull, pituitary gland, or pinealgland. Specific forms of brain cancer that can be treated includeastrocytomas, chondromas, chondrosarcomas, chordomas, CNS (centralnervous system) lymphomas, craniopharyngiomas, ependymomas,gangliogliomas, ganglioneuromas (also called gangliocytomas), gliomas,including astrocytomas, oligodendrogliomas, and ependymomas,hemangioblastomas (also called vascular tumors), primitiveneuroectodermal tumors (PNET) such as medulloblastomas, meningiomas, andvestibular schwannomas (formerly known as acoustic neuroma/schwannoma).

The compounds described herein can also be used to treat metastatictumors that invade the intracranial sphere from cancers originating inother organs of the body. These conditions are typically referred to assecondary brain tumors. Secondary brain tumors that can be treated witha compound described herein include metastatic tumors of the brain thatoriginate from lung cancer, breast cancer, malignant melanoma, kidneycancer, colon cancer, and other carcinomas.

Other examples of cancerous conditions that are within the scope of theinvention include, but are not limited to, neuroblastomas and osteogeniccarcinomas (e.g. cancer of the bone or neoplastic growth of tissue inbone). Examples of malignant primary bone tumors that can be treatedwith a compound described herein include osteosarcomas, chondrosarcomas,Ewing's sarcoma, fibrosarcomas, and the like, and secondary bone tumorssuch as metastatic lesions that have spread from other organs, includingcarcinomas of the breast, lung, and prostate.

The terms alkyl, cycloalkyl, alkenyl, alkenyl, aryl, amino groups,alkoxy, halo, haloalkyl, heteroaryl, heterocycle, and ester are wellknown in the art and have their art-recognized definitions, such asdescribed in U.S. Patent Publication No. 2012/0040995 (Hergenrother etal.).

For example, the term “alkyl” refers to a monoradical branched orunbranched saturated hydrocarbon chain preferably having from 1 to 22carbon atoms and to cycloalkyl groups having one or more rings having 3to 22 carbon atoms. Short alkyl groups are those having 1 to 6 carbonatoms including methyl, ethyl, propyl, butyl, pentyl and hexyl groups,including all isomers thereof. Long alkyl groups are those having 8-22carbon atoms and preferably those having 12-22 carbon atoms as well asthose having 12-20 and those having 16-18 carbon atoms.

Thus, the term “alkyl” can refer to a monoradical branched or unbranchedsaturated hydrocarbon chain preferably having from 1 to 22 carbon atoms.Short alkyl groups are those having 1 to 12 carbon atoms includingmethyl, ethyl, propyl, butyl, pentyl and hexyl groups, including allisomers thereof. Long alkyl groups are those having 12-22 carbon atoms.The group may be a terminal group or a bridging group.

Alkyl, heteroalkyl, aryl, heteroaryl, and heterocycle groups, and cyclicand/or unsaturated versions thereof, can be R groups of Formula I, andeach group can be optionally substituted. Thus, in various embodiments,any one or more of the substituents below can be an R group (e.g., R¹,R², R², R¹⁰, R²⁰, R³⁰, etc.) of a group or formula described herein. Theterm “substituted” indicates that one or more hydrogen atoms on thegroup indicated in the expression using “substituted” is replaced with a“substituent”. The number referred to by ‘one or more’ can be apparentfrom the moiety one which the substituents reside. For example, one ormore can refer to, e.g., 1, 2, 3, 4, 5, or 6; in some embodiments 1, 2,or 3; and in other embodiments 1 or 2. The substituent can be one of aselection of indicated groups, or it can be a suitable group known tothose of skill in the art, provided that the substituted atom's normalvalency is not exceeded, and that the substitution results in a stablecompound. Suitable substituent groups include, e.g., alkyl, alkenyl,alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, aroyl,(aryl)alkyl (e.g., benzyl or phenylethyl), heteroaryl, heterocycle,cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino,trifluoromethyl, trifluoromethoxy, trifluoromethylthio, difluoromethyl,acylamino, nitro, carboxy, carboxyalkyl, keto, thioxo, alkylthio,alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl,heteroarylsulfinyl, heteroarylsulfonyl, heterocyclesulfinyl,heterocyclesulfonyl, phosphate, sulfate, hydroxyl amine,hydroxyl(alkyl)amine, and cyano. Additionally, suitable substituentgroups can be, e.g., —X, —R, —O⁻, —OR, —SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃,—CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃, —NC(═O)R, —C(═O)R,—C(═O)NRR, —S(═O)₂O⁻, —S(═O)₂OH, —S(═O)₂R, —OS(═O)₂OR, —S(═O)₂NR,—S(═O)R, —OP(═O)O₂RR, —P(═O)O₂RR, —P(═O)(O⁻)₂, —P(═O)(OH)₂, —C(═O)R,—C(═O)X, —C(S)R, —C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR,—C(S)NRR, or —C(NR)NRR, where each X is independently a halogen(“halo”): F, Cl, Br, or I; and each R is independently H, alkyl, aryl,(aryl)alkyl (e.g., benzyl), heteroaryl, (heteroaryl)alkyl, heterocycle,heterocycle(alkyl), or a protecting group. As would be readilyunderstood by one skilled in the art, when a substituent is keto (═O) orthioxo (═S), or the like, then two hydrogen atoms on the substitutedatom are replaced. In some embodiments, one or more of the substituentsabove can be excluded from the group of potential values forsubstituents on a substituted group, such as an R group (e.g., R¹, R²,R², R¹⁰, R²⁰, R³⁰, etc.) of a group or formula described herein.

The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to 22carbon atoms having a single cyclic ring or multiple condensed rings.Cycloalkyl grouos include those having 3-8 member rings and those having5 and 6 member rings. Cycloalkyl groups include, by way of example,single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclooctyl, and the like, or multiple ring structures suchas adamantanyl, and the like.

The term “alkenyl” refers to a monoradical of a branched or unbranchedunsaturated hydrocarbon group preferably having from 2 to 22 carbonatoms and to cycloalkenyl groups having one or more rings having 3 to 22carbon atoms wherein at least one ring contains a double bond. Alkenylgroups may contain one or more double bonds (C═C) which may beconjugated. Preferred alkenyl groups are those having 1 or 2 doublebonds. Short alkenyl groups are those having 2 to 6 carbon atomsincluding ethylene (vinyl) propylene, butylene, pentylene and hexylenegroups, including all isomers thereof. Long alkenyl groups are thosehaving 8-22 carbon atoms and preferably those having 12-22 carbon atomsas well as those having 12-20 carbon atoms and those having 16-18 carbonatoms. The term “cycloalkenyl” refers to cyclic alkenyl groups of from 3to 22 carbon atoms having a single cyclic ring or multiple condensedrings in which at least one ring contains a double bond (C═C).Cycloalkenyl groups include, by way of example, single ring structuressuch as cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclooctenyl,cylcooctadienyl and cyclooctatrienyl. The term allyl refers to thealkenyl group CH₂—CH═CH₂.

The term “alkynyl” refers to a monoradical of an unsaturated hydrocarbonpreferably having from 2 to 22 carbon atoms and having one or moretriple bonds (C C). Alkynyl groups include ethynyl, propargyl, and thelike. Short alkynyl groups are those having 2 to 6 carbon atoms,including all isomers thereof. Long alkynyl groups are those having 8-22carbon atoms and preferably those having 12-22 carbon atoms as well asthose having 12-20 carbon atoms and those having 16-18 carbon atoms.

The term “aryl” refers to a group containing an unsaturated aromaticcarbocyclic group of from 6 to 22 carbon atoms having a single ring(e.g., phenyl), one or more rings (e.g., biphenyl) or multiple condensed(fused) rings, wherein at least one ring is aromatic (e.g., naphthyl,dihydrophenanthrenyl, fluorenyl, or anthryl). Aryls include phenyl,naphthyl and the like. Aryl groups may contain portions that are alkyl,alkenyl or akynyl in addition to the the unsaturated aromatic ring(s).The term “alkaryl” refers to the aryl groups containing alkyl portions,i.e., -alkylene-aryl and -substituted alkylene-aryly. Such alkarylgroups are exemplified by benzyl (—CH₂-phenyl), phenethyl and the like.

Alkyl, alkenyl, alkynyl and aryl groups are optionally substituted asdescribed herein (the term(s) can include substituted variations) andmay contain 1-8 non-hydrogen substituents dependent upon the number ofcarbon atoms in the group and the degree of unsaturation of the group.All such variable as described herein can be unsubstituted (in which anyvariables groups that can be hydrogen are hydrogen) or substituted withone or more non-hydrogen substituents selected from halogen, includingfluorine, chlorine, bromine or iodine, C1-C3 haloalkyl, hydroxyl (OH),thiol (HS—), C1-C6 alkyl, C1-C3 alkyl, C1-C6 alkoxy, C1-C3 alkoxy,phenyl, benzyl, alkenyl, C2-C4 alkenyl, alkynyl, C2-C4 alkynyl, —NH₂,—NR′H, —NR′R″, R′CO—, R′R″NCO—, R′CO—NH—, or R′CO—NR′—, where R′ and R″are C1-C6 alkyl, C1-C3 alkyl or phenyl.

The term “amino” refers to the group —NH₂ or to the group NR′ R″ whereeach R′ and R″ is independently selected from the group consisting ofhydrogen, alkyl or aryl groups.

Haloalkyl” refers to alkyl as defined herein substituted by one or morehalo groups as defined herein, which may be the same or different.Representative haloalkyl groups include, by way of example,trifluoromethyl, 3-fluorododecyl, 12,12,12-trifluorododecyl,2-bromooctyl, 3-bromo-6-chloroheptyl, and the like.

The term “heteroaryl” refers to an aromatic group of from 2 to 22 carbonatoms having 1 to 4 heteroatoms selected from oxygen, nitrogen andsulfur within at least one ring (if there is more than one ring).Heteroaryl groups may be optionally substituted. Heteroaryl groupsinclude among others those having 5 and 6-member rings and those havingone or two nitrogens in the ring, those having one or two oxygens in thering as well as those having one or two sulfurs in the ring.

The term “heterocycle” or “heterocyclic” refers to a monoradicalsaturated or unsaturated group having a single ring or multiplecondensed rings, from 2-22 carbon atoms and from 1 to 6 hetero atoms,preferably 1 to 4 heteroatoms, selected from nitrogen, sulfur,phosphorus, and/or oxygen within at least one ring. Heterocyclic groupsmay be substituted. Rings preferably have 3-10 members and morespecifically have 5 or 6 members.

The term “ester” refers to chemical entities as understood in the artand in particular can include groups of the form (RCO—).

The compounds of this invention include all novel stereochemical isomersarising from the substitution of disclosed compounds.

Pharmaceutical Formulations

The compounds described herein can be used to prepare therapeuticpharmaceutical compositions. The compounds may be added to thecompositions in the form of a salt or solvate. For example, in caseswhere compounds are sufficiently basic or acidic to form stable nontoxicacid or base salts, administration of the compounds as salts may beappropriate. Examples of pharmaceutically acceptable salts are organicacid addition salts formed with acids that form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartrate, succinate, benzoate, ascorbate,α-ketoglutarate, and β-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, halide, sulfate, nitrate,bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid to provide aphysiologically acceptable ionic compound. Alkali metal (for example,sodium, potassium or lithium) or alkaline earth metal (for example,calcium) salts of carboxylic acids can also be prepared by analogousmethods.

The compounds of the formulas described herein can be formulated aspharmaceutical compositions and administered to a mammalian host, suchas a human patient, in a variety of forms. The forms can be specificallyadapted to a chosen route of administration, e.g., oral or parenteraladministration, by intravenous, intramuscular, topical or subcutaneousroutes.

The compounds described herein may be systemically administered incombination with a pharmaceutically acceptable vehicle, such as an inertdiluent or an assimilable edible carrier. For oral administration,compounds can be enclosed in hard or soft shell gelatin capsules,compressed into tablets, or incorporated directly into the food of apatient's diet. Compounds may also be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations typically contain at least 0.1% ofactive compound. The percentage of the compositions and preparations canvary and may conveniently be from about 2% to about 60% of the weight ofa given unit dosage form. The amount of active compound in suchtherapeutically useful compositions is such that an effective dosagelevel can be obtained.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; and a lubricant such as magnesium stearate. A sweeteningagent such as sucrose, fructose, lactose or aspartame; or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring, maybe added. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweetening agent, methyl and propyl parabens as preservatives, a dye andflavoring such as cherry or orange flavor. Any material used inpreparing any unit dosage form should be pharmaceutically acceptable andsubstantially non-toxic in the amounts employed. In addition, the activecompound may be incorporated into sustained-release preparations anddevices.

The active compound may be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can be prepared in glycerol, liquidpolyethylene glycols, triacetin, or mixtures thereof, or in apharmaceutically acceptable oil. Under ordinary conditions of storageand use, preparations may contain a preservative to prevent the growthof microorganisms.

Pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions, dispersions, or sterile powderscomprising the active ingredient adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. The ultimate dosage form should besterile, fluid and stable under the conditions of manufacture andstorage. The liquid carrier or vehicle can be a solvent or liquiddispersion medium comprising, for example, water, ethanol, a polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycols, andthe like), vegetable oils, nontoxic glyceryl esters, and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe formation of liposomes, by the maintenance of the required particlesize in the case of dispersions, or by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thiomersal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, buffers, or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by agents delayingabsorption, for example, aluminum monostearate and/or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation can include vacuumdrying and freeze drying techniques, which yield a powder of the activeingredient plus any additional desired ingredient present in thepreviously sterile-filtered solutions.

For topical administration, compounds may be applied in pure form, e.g.,when they are liquids. However, it will generally be desirable toadminister the active agent to the skin as a composition or formulation,for example, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina, and the like. Useful liquidcarriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, orwater-alcohol/glycol blends, in which a compound can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed onto the affectedarea using a pump-type or aerosol sprayer.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses, or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of dermatological compositions for delivering active agents tothe skin are known to the art; for example, see U.S. Pat. No. 4,992,478(Geria), U.S. Pat. No. 4,820,508 (Wortzman), U.S. Pat. No. 4,608,392(Jacquet et al.), and U.S. Pat. No. 4,559,157 (Smith et al.). Suchdermatological compositions can be used in combinations with thecompounds described herein where an ingredient of such compositions canoptionally be replaced by a compound described herein, or a compounddescribed herein can be added to the composition

Useful dosages of the compounds described herein can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949 (Borch et al.). The amount of a compound, or anactive salt or derivative thereof, required for use in treatment willvary not only with the particular compound or salt selected but alsowith the route of administration, the nature of the condition beingtreated, and the age and condition of the patient, and will beultimately at the discretion of an attendant physician or clinician.

The compound can be conveniently administered in a unit dosage form, forexample, containing 5 to 1000 mg/m², conveniently 10 to 750 mg/m², mostconveniently, 50 to 500 mg/m² of active ingredient per unit dosage form.The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

The compounds described herein can be effective anti-tumor agents andhave higher potency and/or reduced toxicity as compared to PAC-1.Compounds of the invention can be more potent and less toxic than PAC-1,and/or avoid a potential site of catabolic metabolism encountered withPAC-1, i.e., they have a different metabolic profile than PAC-1.

The invention provides therapeutic methods of treating cancer in amammal, which involve administering to a mammal having cancer aneffective amount of a compound or composition described herein. A mammalincludes a primate, human, rodent, canine, feline, bovine, ovine,equine, swine, caprine, bovine and the like. Cancer refers to anyvarious type of malignant neoplasm, for example, colon cancer, breastcancer, melanoma and leukemia, and in general is characterized by anundesirable cellular proliferation, e.g., unregulated growth, lack ofdifferentiation, local tissue invasion, and metastasis.

The ability of a compound of the invention to treat cancer may bedetermined by using assays well known to the art. For example, thedesign of treatment protocols, toxicity evaluation, data analysis,quantification of tumor cell kill, and the biological significance ofthe use of transplantable tumor screens are known. In addition, abilityof a compound to treat cancer may be determined using the tests asdescribed below.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention.

EXAMPLES Example 1 General Procedure for the Synthesis of PAC-1Analogues

To a 16×150 mm test tube were added hydrazide (1.7 equiv), aldehyde (1.0equiv), 2-ethoxyethanol (1 mL), and 1.2 M HCl (10 mol %). The tubes wereshaken on a Man Syncore parallel synthesizer at 110° C. until allaldehyde had reacted (as monitored by ESI-MS). The reaction mixture wascooled to room temperature (˜23° C.), and polystyrene-benzaldehyde (3.5equiv) was added. The reaction mixture was shaken at 25-80° C. until nohydrazide remained (as monitored by ESI-MS). The reaction mixture wascooled to room temperature, and the resin was filtered and washed with2-ethoxyethanol. The filtrate was dried under high vacuum to afford thePAC-1 analogue.

Example 2 Parallel Synthesis of PAC-1 Analogues

Reactions requiring anhydrous conditions were conducted under a positiveatmosphere of nitrogen or argon in oven-dried glassware. Standardsyringe techniques were used for anhydrous addition of liquids. Unlessotherwise noted, all starting materials, solvents, and reagents wereacquired from commercial suppliers and used without furtherpurification. Flash chromatography was performed using 230-400 meshsilica gel. Compounds 1{1}, 1{2}, 1{3}, 1{4}, 1{5}, 1{6}, 2{24}, 2{25},2{26}, and PAC-1 were synthesized as previously reported.

Compound Analysis.

NMR experiments were recorded either in CDCl₃ (Sigma), CD₃OD (Sigma) or(CD₃)₂CO (Sigma) on a Varian Unity 400 MHz or 500 MHz spectrometer withresidual undeuterated solvent as the internal reference for ¹H-NMR and¹³C-NMR experiments and 1% CFCl₃/CDCl₃ as the external reference for¹⁹F-NMR experiments. Chemical shift, 6 (ppm); coupling constants, J(Hz); multiplicity (s=singlet, d=doublet, t=triplet, q=quartet,m=multiplet, br=broad); and integration are reported. High-resolutionmass spectral data was recorded on a Micromass Q-Tof Ultima hybridquadrupole/time-of-flight ESI mass spectrometer at the University ofIllinois Mass Spectrometry Laboratory.

General Procedure A: Synthesis of 1-benzylpiperazines.

Anhydrous piperazine (6.0 equiv.) was suspended in THF (0.45 M benzylhalide). The mixture was heated to reflux until piperazine fullydissolved. Upon dissolution, the substituted benzyl halide (1.0 equiv.)was added to the reaction mixture. A white solid immediately formed. Thereaction mixture was stirred at reflux for 2.5 hours. The mixture wascooled to room temperature. The solid was filtered and washed with THFand EtOAc. The combined filtrates were concentrated to 10% of theoriginal volume. The concentrate was poured into a separatory funnelwith 5% brine/H₂O made basic (pH>12) with KOH. The aqueous layer wasextracted with DCM and EtOAc. The organic layers were combined, driedover Na₂SO₄, and concentrated. The crude product was purified by silicagel column chromatography to yield pure 1-benzylpiperazine.

General Procedure B: Synthesis of Ethyl Esters.

The substituted 1-benzylpiperazine (1.0 equiv.) was dissolved in acetone(0.5 M), and chloroform was added to some reaction mixtures as needed tofully dissolve the 1-benzylpiperazine. NaHCO₃ (1.25 equiv.) was added,and the mixture was stirred at room temperature. Ethyl chloroacetate(1.1 equiv.) was then added dropwise. The reaction mixture was stirredovernight at reflux. The reaction mixture was cooled to roomtemperature. The solid was filtered and washed with acetone. Thefiltrate was concentrated. The crude product was purified by silica gelcolumn chromatography to yield pure ethyl ester.

General Procedure C: Synthesis of Hydrazides.

The substituted ethyl 2-(4-benzylpiperazin-1-yl)acetate (1.0 equiv.) wasdissolved in EtOH (0.5 M). The solution was stirred, and anhydroushydrazine (3.0-4.0 equiv.) was added dropwise. The reaction mixture wasstirred at reflux overnight. The reaction mixture was cooled to roomtemperature and concentrated. The concentrate was transferred to aseparatory funnel containing 1:1 brine:H₂O made basic (pH>12) with KOH.The aqueous layer was extracted with DCM (3×) and EtOAc (1×). Thecombined organic layers were dried over MgSO₄ and concentrated. Thecrude product was purified by silica gel column chromatography orrecrystallization to yield pure hydrazide.

The corresponding 1-benzoylpiperazines can be prepared by analogousmethods, for example, as described and illustrated herein, usingappropriate optionally substituted benzoyl halides or benzoic acids, andamide forming conditions such as an amine base (e.g., Et₃N) or EDC-HCland DMAP.

Various useful preparatory methods are described in U.S. PatentPublication Nos. 2011/0257398 (Hergenrother et al.), 2012/0040995(Hergenrother et al.), and 2013/0096133 (Hergenrother et al.). Theinvention is also directed to compounds described in the aforementionedpublications where the piperazine nitrogen distal to the hydrazidemoiety is connected to a carbonyl as opposed to a methylene.

One example of a general synthetic procedure is described below.

1-(4-(tert-butyl)benzyl)piperazine (5 {20})

Synthesized according to General Procedure A: 4-tert-butylbenzyl bromide(4 {20}, 4.05 mL, 22.0 mmol, 1 equiv.), piperazine (11.4 g, 132.1 mmol,6 equiv.), THF (48.1 mL). Purification with flash column chromatographyon silica gel (4:1 EtOAc:MeOH) afforded 5{20} (3.75 g, 73%) as a beigesolid. ¹H-NMR (500 MHz, CDCl₃): δ 7.30 (d, 2H, J=8.5 Hz), 7.22 (d, 2H,J=8.5 Hz), 3.44 (s, 2H), 2.85 (t, 4H, J=5.0 Hz), 2.38 (br s, 4H), 1.54(br s, 1H), 1.29 (s, 9H). ¹³C-NMR (125 MHz, CDCl₃): δ 149.6, 134.7,128.7, 124.8, 63.1, 54.3, 45.9, 34.2, 31.2.

Ethyl 2-(4-(4-(tert-butyl)benzyl)piperazin-1-yl)acetate (6{20})

Synthesized according to General Procedure B: 5{20} (3.46 g, 14.9 mmol,1 equiv.), ethyl chloroacetate (1.8 mL, 16.4 mmol, 1.1 equiv.), NaHCO₃(1.56 g, 18.6 mmol, 1.25 equiv.), acetone (29.8 mL), chloroform (10 mL).Purification with flash column chromatography on silica gel (1:1hexanes:EtOAc) afforded 6{20} (4.25 g, 90%) as an orange oil. ¹H-NMR(500 MHz, CDCl₃): δ 7.31 (d, 2H, J=8.5 Hz), 7.21 (d, 2H, J=8.5 Hz), 4.16(q, 2H, J=7.0 Hz), 3.48 (s, 2H), 3.18 (s, 2H), 2.58 (br s, 4H), 2.51 (brs, 4H), 1.29 (s, 9H), 1.24 (t, 3H, J=7.0 Hz). ¹³C-NMR (125 MHz, CDCl₃):δ 170.1, 149.7, 134.7, 128.8, 124.9, 62.5, 60.4, 59.4, 53.0, 52.6, 34.3,31.3, 14.1.

2-(4-(4-(tert-butyl)benzyl)piperazin-1-yl)acetohydrazide (1{20})

Synthesized according to General Procedure C: 6{20} (4.12 g, 12.9 mmol,1 equiv.), anhydrous hydrazine (1.2 mL, 38.8 mmol, 3 equiv.), ethanol(26.0 mL). Purification by silica gel column chromatography (4:1EtOAc:MeOH) afforded 1{20} (3.36 g, 84%) as a beige solid. ¹H-NMR (500MHz, CDCl₃): δ 8.17 (br s, 1H), 7.31 (d, 2H, J=8.5 Hz), 7.20 (d, 2H,J=8.5 Hz), 3.85 (br s, 2H), 3.46 (s, 2H), 3.05 (s, 2H), 2.52 (br s, 4H),2.45 (br s, 4H), 1.30 (s, 9H). ¹³C-NMR (125 MHz, CDCl₃): δ 170.4, 149.9,134.5, 128.7, 125.0, 62.4, 60.5, 53.5, 52.9, 34.3, 31.3.

N′-(3-allyl-2-hydroxybenzylidene)-2-(4-(4-(tert-butyl)benzyl)piperazin-1-yl)acetohydrazide(3{20,24})

To a stirred solution of hydrazide 1{20} (100 mg, 0.33 mmol, 1.0 equiv.)and aldehyde 2{24} (53.5 mg, 0.33 mmol, 1.0 equiv.) in EtOH (2.2 mL,0.15 M) was added 1.2 M HCl (7 mol %). The reaction mixture was stirredat reflux overnight. The reaction mixture was cooled to room temperatureand concentrated. The crude product was purified by silica gel columnchromatography (gradient, 0-10% MeOH/EtOAc) to yield 3{20,24} (102.0 mg,0.23 mmol, 68.9%) as a light brown solid. ¹H-NMR (500 MHz, CDCl₃): δ11.31 (br s, 1H), 10.09 (br s, 1H), 8.38 (s, 1H), 7.36 (d, 2H, J=8.5Hz), 7.25 (d, 2H, J=8.5 Hz), 7.19 (d, 1H, J=7.5 Hz), 7.08 (dd, 1H,J=1.5, 7.5 Hz), 6.85 (t, 1H, J=7.5 Hz), 6.04 (tdd, 1H, J=6.5, 10.0, 16.5Hz), 5.12-5.06 (m, 2H), 3.52 (s, 2H), 3.47 (d, 2H, J=6.5 Hz), 3.20 (s,2H), 2.63 (br s, 4H), 2.54 (br s, 4H), 1.33 (s, 9H). ¹³C-NMR (125 MHz,CDCl₃): δ 165.8, 156.3, 151.1, 150.0, 136.4, 134.6, 132.2, 129.1, 128.7,128.1, 125.1, 118.9, 116.8, 115.6, 62.5, 60.9, 53.6, 53.0, 34.4, 33.8,31.3. HRMS (ESI): 449.2915 (M+1); calcd. for C₂₇H₃₇N₄O₂: 449.2917.

Example 3 Biological Evaluation of Analogues of PAC-1

Materials.

All reagents were obtained from Fisher unless otherwise indicated. Allbuffers were made with MilliQ purified water. Ac-DEVD-pNA wassynthesized as previously described.⁵ Luria broth (LB) was obtained fromEMD. Doxorubicin was obtained from Sigma. Caspase Activity Buffercontains 50 mM HEPES, 300 mM NaCl, 1.5 mM TCEP, 0.01% TritonX-100 and isChelex® treated. Ni NTA Binding Buffer contains 50 mM Tris (pH 8.0), 300mM NaCl, and 10 mM imidazole. Ni NTA Wash Buffer contains 50 mM Tris (pH8.0), 300 mM NaCl, and 50 mM imidazole. Ni NTA Elution Buffer contains50 mM Tris (pH 8.0), 300 mM NaCl, and 500 mM imidazole. Annexin VBinding Buffer contains 10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2,0.1% BSA. The C-terminal 6×His-tagged procaspase-3 proteins wereexpressed as described below.

Cell Culture.

U-937 cells were obtained from the American Type Culture Collection andmaintained at low passage number. Cultures were maintained in RPMI 1640supplemented with 10% fetal bovine serum and 1% penicillin-streptomycinand grown at 37° C. and 5% CO₂.

Cell Death Assay for Initial Screen.

Compound (2 μL of a 10 mM DMSO solution) was added in singlet by directaddition to a well containing 998 μL U-937 cells (1×10⁶) in RPMI 1640media (10% FBS) at a final compound concentration of 20 μM. Afterincubation at 37° C. for 24 h, the cells were transferred to flowcytometry tubes, washed, and resuspended in Annexin V binding buffer.The cells were double stained with FITC-Annexin V and propidium iodideand a cell population of at least 10,000 events was collected by the LSRII flow cytometer. Percent viable cells (Annexin V—negative, propidiumiodide—negative) were determined using FCS Express software.

72 Hour IC₅₀ Cell Death Assay.

U-937 human lymphoma cells were plated into the wells of 96 well plateat a density of 15,000 cells per well in 99 μL of RPMI 1640 growth mediawith 10% FBS and 1% pen-strep. To each well was added 1 μL of compoundstock solutions in DMSO at varying concentrations such that the cellswere treated with concentrations between 0 μM and 100 μM compound. Eachconcentration was tested in quintuplicate per plate. In each plate 5wells received 20 μM doxorubicin as a positive death control and 5 wellsreceived 1 μL of DMSO as a negative control. The plates were thenincubated at 37° C. and 5% CO2 for 72 hours.

After the 72 hour incubation period, the plates were analyzed using aSulforhodamine B assay (Vichai and Kirtikara, Nat Protoc 2006, 1,1112-1116). Specifically, to each well of the plate 25 μL of a 50% (w/v)solution of trichloroacetic acid in H₂O was added and the plates wereincubated for 4 hours at 4° C. The plates were then washed gently withwater five times. The plates were allowed to air dry after which 100 μLof a 0.057% (w/v) Sulforhodamine B in a 1% (v/v) acetic acid solutionwas added to each well for 30 minutes at room temperature. The plateswere gently washed 5 times with 1% (v/v) acetic acid and air dried. 200μL of 10 mM Tris base (pH 10.4) was added to each well and the plateswere placed on an orbital shaker for thirty minutes. The level of SRBwas quantified fluorometrically at excitation and emission wavelengths488 and 585 nm, respectively, on a Molecular Devices plate reader andthe percent cell death calculated and normalized to the positive control(100% cell death) and the negative control (0% cell death). The percentcell death was averaged for each compound concentration and plotted as afunction of compound concentration. The data were fit to a logisticaldose response curve using Table curve 2D and the IC₅₀ value wascalculated. The experiment was repeated three times and the average ofthe calculated IC₅₀ values was reported. The standard error of the mean(SEM) was determined and reported for the triplicate experiments.

Induction of Apoptosis by Hit Compounds.

U-937 Cells (1 mL of 6×10⁵ cells/mL) were treated with 10 μL of 750 μMDMSO solutions of the compounds to achieve a final concentration of 7.5μM. The cells were incubated at 37° C. for 24 hours. The cells wereharvested via centrifugation (200 g for 5 minutes), washed with PBS (2mL), and resuspended in 450 μL Annexin V Binding Buffer. To each samplewas added 3.5 μL of FITC conjugated Annexin V stain (Southern Biotech)and 3.5 μL of propidium iodide (Sigma) to a final concentration of 50μg/mL. Cell populations were analyzed on a Benton Dickinson LSR II cellflow cytometer.

Recombinant Expression and Purification of Procaspase-3.

Techniques adapted from Hergenrother and coworkers (Putt et al., NatChem Biol 2006, 2, 543-550). A 20 mL volume of an overnight culture ofRosetta E. coli containing the procaspase-3 (wild-type) expressionplasmid was seeded into 2 L of LB media containing ampicillin. Theculture was grown to an OD600=1.0, at which point protein expression wasinduced via addition of IPTG (to 1 mM); the culture was allowed to growfor 30 additional minutes. Cells were then harvested (10 minute spins at10,000×g and re-suspended in NTA binding buffer (300 mM NaCl, 50 mMTris, 10 mM imidazole, pH 8.0). The cells were lysed on ice viasonication. The cell lysate was then spun at 35,000×g for 35 mM Thesupernatant was decanted and 1 mL of nickel-NTA resin was added. Thecell lysate was incubated for 45 minutes at 4° C. The resin was loadedon a column, washed with 10 mL NTA binding buffer followed by 10 mL NTAwash buffer (300 mM NaCl, 50 mM Tris, 50 mM imidazole, pH 8.0). Theproteins were eluted in 0.5 mL fractions with 10 mL of NTA elutionbuffer (300 mM NaCl, 50 mM Tris, 500 mM imidazole, pH 8.0). Fractionscontaining protein were pooled and further purified to remove anycontaminating zinc by applying the protein to a PD-10 column (GEHealthcare) charged with Caspase Activity Buffer that had been treatedwith Chelex® resin. The resulting concentration was determined using theEdelhoch method and the molar absorptivity of procaspase-3 of 26150 M⁻¹cm⁻¹. Protein stocks were flash-frozen in liquid nitrogen and stored at−80° C.

Procaspase-3 Activity Assay.

In a 384-well plate recombinantly expressed, zinc-free procaspase-3(wild type, at 1 μM) in Caspase Activity Buffer (50 mM HEPES, 300 mMNaCl, 1.5 mM TCEP, 0.01% TritonX-100) was incubated at 37° C. in thepresence of 3.5 μM ZnSO₄ and the basal activity was assessed via theaddition of Ac-DEVD-pNA (final concentration in well of 200 μM). Theabsorbance at 405 nm was monitored with a SpectraMax plate reader(Molecular Devices). After the basal activity was determined, DMSO,PAC-1 and analogues were added to each sample to a final concentrationof 25 μM. Activity of each treatment was assessed at 5, 20, 40 and 60minutes via 5-minute kinetic reads. The slope of each data set was usedto determine the activity of the protein. Activity was normalized to apercent activity at each time point using a zinc-free sample and azinc-inhibited sample treated with DMSO.

Example 4 Compound Preparation

Materials and Methods.

General

All reactions requiring anhydrous conditions were conducted under apositive atmosphere of nitrogen or argon in oven-dried glassware.

Standard syringe techniques were used for anhydrous addition of liquids.Unless otherwise noted, all starting materials, solvents, and reagentswere acquired from commercial suppliers and used without furtherpurification. Flash chromatography was performed using 230-400 meshsilica gel. Syntheses of 46a,¹ 46 b,² 46 h,³ 47 a,² 50,² PAC-1 (1),¹ andS-PAC-1 (2)² have been described previously.

Compound Analysis.

NMR experiments were recorded either in CDCl₃ (Sigma or Cambridge),CD₃OD (Sigma), or (CD₃)₂C0 (Sigma or Cambridge) on a Varian Unity 500MHz spectrometer with residual undeuterated solvent as the internalreference for ¹H-NMR and ¹³C-NMR, and C₆F₆ as the internal reference for¹⁹F-NMR. Chemical shift, 6 (ppm); coupling constants, J (Hz);multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, quint=quintet,sext=sextet, m=multiplet, br=broad); and integration are reported.High-resolution mass spectral data was recorded on a Micromass Q-TofUltima hybrid quadrupole/time-of-flight ESI mass spectrometer or aMicromass 70-VSE at the University of Illinois Mass SpectrometryLaboratory.

General Procedure A: Synthesis of Dialkylated Piperazines.

To a round-bottom flask were added benzyl halide (1.0 equiv.), K₂CO₃(3.0 equiv.), and acetone (0.2 M). The mixture was stirred, and 50 (1.5equiv.) was added. The reaction mixture was stirred at reflux overnight.The reaction mixture was cooled to room temperature. The solid wasfiltered and washed with acetone. The filtrate was concentrated, and theproduct was purified by silica gel column chromatography.

General Procedure B: Synthesis of Amides.

To an oven-dried round-bottom flask were added 50 (1.0 equiv.),anhydrous tetrahydrofuran (0.2 M), and freshly distilled Et₃N (2.0equiv.). The solution was stirred at 0° C. under N₂, and the benzoylchloride (1.0 equiv.) was added. The reaction mixture was stirredovernight at room temperature under N₂. The reaction mixture was dilutedwith EtOAc and washed with sat. NaHCO₃ (2×), H₂O, and brine. The organiclayer was dried over MgSO₄, filtered, and concentrated. The product waspurified by silica gel column chromatography.

General Procedure C: Synthesis of Hydrazides.

To a round-bottom flask were added ethyl ester (1.0 equiv.) and EtOH or2:1 EtOH:MeOH (0.5 M). The solution was stirred, and anhydrous hydrazine(4.0 equiv.) was added dropwise. The reaction mixture was stirred atreflux overnight. The reaction mixture was cooled to room temperatureand concentrated. The resulting residue was partitioned betweenCH₂Cl₂/1:1 brine:0.1 M KOH. The layers were separated, and the aqueouslayer was extracted with CH₂Cl₂ (2×). The combined organic layers weredried over MgSO₄, filtered, and concentrated. Purification by silica gelcolumn chromatography or recrystallization yielded pure hydrazide.

Ethyl 2-(4-benzoylpiperazin-1-yl)acetate (51c)

Synthesized according to General Procedure B: 50 (2.45 g, 14.2 mmol, 1.0equiv.), anhydrous tetrahydrofuran (70 mL, 0.2 M), freshly distilledEt₃N (4.0 mL, 28.4 mmol, 2.0 equiv.), benzoyl chloride (54c, 2.0 g, 1.7mL, 1.0 equiv.). Purification by silica-gel column chromatography(50-100% EtOAc/hexanes) afforded 51c (2.87 g, 73.1%) as a pale yellowoil. ¹H-NMR (500 MHz, CDCl₃) δ 7.41-7.38 (m, 5H), 4.19 (q, 2H, J=7.0Hz), 3.85 (br s, 2H), 3.48 (br s, 2H), 3.25 (s, 2H), 2.68 (br s), 2.54(br s, 2H), 1.27 (t, 3H, J=7.0 Hz). ¹³C-NMR (125 MHz, CDCl₃) δ 170.5,170.2, 135.9, 129.9, 128.7, 127.3, 61.0, 59.4, 53.3 (br), 52.8 (br),47.8 (br), 42.1 (br), 14.4. HRMS (ESI): 277.1552 (M+1); calcd. forC₁₅H₂₁N₂O₃: 277.1552.

2-(4-benzoylpiperazin-1-yl)acetohydrazide (46c)

Synthesized according to General Procedure C: 51c (2.87 g, 10.4 mmol,1.0 equiv.), anhydrous hydrazine (1.31 mL, 41.6 mmol, 4.0 equiv.), EtOH(20 mL, 0.5 M). 46c (1.41 g, 51.5%) was obtained as a white solid afterextraction without further purification. ¹H-NMR (500 MHz, CDCl₃) δ 8.10(s, 1H), 7.39-7.34 (m, 5H), 3.84 (br s, 2H), 3.77 (br s, 2H), 3.43 (brs, 2H), 3.08 (s, 2H), 2.56 (br s, 2H), 2.44 (br s, 2H). ¹³C-NMR (125MHz, CDCl₃) δ 170.5, 169.9, 135.5, 130.0, 128.7, 127.1, 60.6, 53.9 (br),53.4 (br), 47.7 (br), 42.2 (br). HRMS (ESI): 263.1513 (M+1); calcd. forC₁₃H₁₉N₄O₂: 263.1508.

Ethyl 2-(4-(4-cyanobenzyl)piperazin-1-yl)acetate (51d)

Synthesized according to General Procedure A:4-(bromomethyl)benzonitrile (54d, 2.0 g, 10.2 mmol, 1.0 equiv.), 50(2.64 g, 15.3 mmol, 1.5 equiv.), K₂CO₃ (4.22 g, 30.6 mmol, 3.0 equiv.),acetone (50 mL, 0.2 M). Purification by silica gel column chromatography(50-100% EtOAc/hexanes) afforded 51d (2.71 g, 92.3%) as a yellow solid.¹H-NMR (500 MHz, CDCl₃) δ 7.60 (d, 2H, J=8.0 Hz), 7.44 (d, 2H, J=8.0Hz), 4.18 (q, 2H, J=7.0 Hz), 3.55 (s, 2H), 3.20 (s, 2H), 2.61 (br s,4H), 2.51 (br s, 4H), 1.26 (t, 3H, J=7.0 Hz). ¹³C-NMR (125 MHz, CDCl₃) δ170.4, 144.4, 132.3, 129.7, 119.2, 111.0, 62.5, 60.8, 59.6, 53.1, 53.1,14.4. HRMS (ESI): 288.1718 (M+1); calcd. for C₁₆H₂₂N₃O₂: 288.1712.

2-(4-(4-cyanobenzyl)piperazin-1-yl)acetohydrazide (46d)

Synthesized according to General Procedure C: 51d (2.71 g, 9.43 mmol,1.0 equiv.), anhydrous hydrazine (1.18 mL, 37.7 mmol, 4.0 equiv.), EtOH(19 mL, 0.5 M). 46d (1.73 g, 67.1%) was obtained as an off-white solidafter extraction without further purification. ¹H-NMR (500 MHz, CDCl₃) δ8.10 (br s, 1H), 7.60 (d, 2H, J=8.0 Hz), 7.43 (d, 2H, J=8.5 Hz), 3.84(br d, 2H, J=5.0 Hz), 3.55 (s, 2H), 3.08 (s, 2H), 2.55 (br s, 4H), 2.46(br s, 4H). ¹³C-NMR (125 MHz, CDCl₃) δ 170.6, 144.1, 132.4, 129.6,119.1, 111.2, 62.5, 60.8, 53.8, 53.3. HRMS (ESI): 274.1673 (M+1); calcd.for C₁₄H₂₀N₅O: 274.1668.

Ethyl 2-(4-(4-cyanobenzoyl)piperazin-1-yl)acetate (51e)

Synthesized according to General Procedure B: 50 (5.20 g, 30.2 mmol, 1.0equiv.), anhydrous tetrahydrofuran (150 mL, 0.2 M), freshly distilledEt₃N (8.4 mL, 60.4 mmol, 2.0 equiv.), 4-cyanobenzoyl chloride (54e, 5.0g, 30.2 mmol, 1.0 equiv.). Purification by silica gel columnchromatography (0-10% MeOH/EtOAc) afforded 51e (5.95 g, 65.4%) as awhite solid. ¹H-NMR (500 MHz, CDCl₃) δ 7.68 (d, 2H, J=8.0 Hz), 7.47 (d,2H, J=8.0 Hz), 4.14 (q, 2H, J=7.0 Hz), 3.80 (br s, 2H), 3.37 (br s, 2H),3.23 (s, 2H), 2.67 (br s, 2H), 2.53 (br s, 2H), 1.23 (t, 3H, J=7.0 Hz).¹³C-NMR (125 MHz, CDCl₃) δ 170.0, 168.3, 140.1, 132.5, 127.9, 118.2,113.6, 60.9, 59.0, 52.9, 52.3, 47.5, 42.1, 14.3. HRMS (ESI): 302.1501(M+1); calcd. for C₁₆H₂₀N₃O₃: 302.1505.

2-(4-(4-cyanobenzoyl)piperazin-1-yl)acetohydrazide (46e)

Synthesized according to General Procedure C with modification as noted:51e (5.59 g, 18.6 mmol, 1.0 equiv.), anhydrous hydrazine (2.4 mL, 74.4mmol, 4.0 equiv.), EtOH (35 mL, 0.5 M). After extraction with CH₂Cl₂,the aqueous layer was extracted with EtOAc (3×). 46e (2.98 g, 55.8%) wasobtained as an off-white solid after extraction without furtherpurification. ¹H-NMR (500 MHz, CDCl₃) δ 8.02 (br s, 1H), 7.70 (d, 2H,J=8.5 Hz), 7.48 (d, 2H, J=8.5 Hz), 3.86 (br d, 2H, J=3.5 Hz), 3.78 (brs, 2H), 3.37 (br s, 2H), 3.11 (s, 2H), 2.60 (br s, 2H), 2.46 (br s, 2H).¹³C-NMR (125 MHz, CDCl₃) δ 169.8, 168.4, 139.8, 132.6, 127.9, 118.1,113.8, 60.6, 53.8 (br), 53.3 (br), 47.6 (br), 42.2 (br). HRMS (ESI):288.1464 (M+1); calcd. for C₁₄H₁₈N₅O₂: 288.1461.

Ethyl 2-(4-(4-fluorobenzyl)piperazin-1-yl)acetate (51f)

Synthesized according to General Procedure A: 4-fluorobenzyl chloride(54f, 2.5 g, 2.1 mL, 17.3 mmol, 1.0 equiv.), 50 (4.48 g, 26.0 mmol, 1.5equiv.), K₂CO₃ (7.19 g, 52.0 mmol, 3.0 equiv.), acetone (90 mL, 0.2 M).Purification by silica gel column chromatography (gradient, 50-100%EtOAc/hexanes) afforded 51f (3.66 g, 75.4%) as a yellow oil. ¹H-NMR (500MHz, CDCl₃) δ 7.24-7.20 (m, 2H), 6.95-6.91 (m, 2H), 4.13 (q, 2H, J=7.0Hz), 3.42 (s, 2H), 3.15 (s, 2H), 2.55 (br s, 4H), 2.46 (br s, 4H), 1.21(t, 3H, J=7.0 Hz). ¹³C-NMR (125 MHz, CDCl₃) δ 170.3, 162.0 (d,J_(C-F)=243.5 Hz), 133.9, 130.6 (d, J_(C-F)=8.0 Hz), 115.0 (d,J_(C-F)=21.0 Hz), 62.2, 60.6, 59.6, 53.1, 52.8, 14.3. ¹⁹F-NMR (470 MHz,CDCl₃) δ −119.1. HRMS (ESI): 281.1659 (M+1); calcd. for C₁₅H₂₂FN₂O₂:281.1665.

2-(4-(4-fluorobenzyl)piperazin-1-yl)acetohydrazide (46f)

Synthesized according to General Procedure C: 51f (3.0 g, 10.7 mmol, 1.0equiv.), anhydrous hydrazine (1.4 mL, 42.8 mmol, 4.0 equiv.), EtOH (20mL, 0.5 M). 46f (2.59 g, 91.1%) was obtained as a white solid afterextraction without further purification. ¹H-NMR (500 MHz, CDCl₃) δ 8.15(br s, 1H), 7.22-7.19 (m, 2H), 6.95-6.91 (m, 2H), 3.84 (br s, 2H), 3.40(s, 2H), 3.01 (s, 2H), 2.47 (br s, 4H), 2.39 (br s, 4H). ¹³C-NMR (125MHz, CDCl₃) δ 170.5, 162.0 (d, J_(C-F)=243.6 Hz), 133.7 (d, J_(C-F)=2.8Hz), 130.6 (d, J_(C-F)=8.3 Hz), 115.1 (d, J_(C-F)=21.1 Hz), 62.0, 60.6,53.7, 53.0. ¹⁹F-NMR (470 MHz, CDCl₃) δ −118.9. HRMS (ESI): 267.1630(M+1); calcd. for C₁₃H₂₀FN₄O: 267.1621.

Ethyl 2-(4-(4-fluorobenzoyl)piperazin-1-yl)acetate (51g)

Synthesized according to General Procedure B: 50 (2.58 g, 15.0 mmol, 1.0equiv.), anhydrous tetrahydrofuran (30 mL, 0.5 M), freshly distilledEt₃N (4.2 mL, 30.0 mmol, 2.0 equiv.), 4-fluorobenzoyl chloride (54 g,1.8 mL, 15.0 mmol, 1.0 equiv.). Purification by silica gel columnchromatography (50-100% EtOAc/hexanes) afforded 51g (3.74 g, 84.7%) as apale yellow oil. ¹H-NMR (500 MHz, CDCl₃) δ ¹H-NMR (500 MHz, CDCl₃) δ7.38-7.34 (m, 2H), 7.06-7.01 (m, 2H), 4.13 (q, 2H, J=7.0 Hz), 3.77 (brs, 2H), 3.43 (br s, 2H), 3.21 (s, 2H), 2.61 (br s, 2H), 2.52 (br s, 2H),1.22 (t, 3H, J=7.0 Hz). ¹³C-NMR (125 MHz, CDCl₃) δ 170.0, 169.4, 163.5(d, J_(C-F)=248.1 Hz), 131.8, 129.5 (d, J_(C-F)=8.3 Hz), 115.6 (d,J_(C-F)=22.0 Hz), 60.8, 59.1, 52.8 (br), 47.8 (br), 42.2 (br), 14.3.¹⁹F-NMR (470 MHz, CDCl₃) δ −113.4. HRMS (ESI): 295.1457 (M+1); calcd.for C₁₅H₂₀FN₂O₃: 295.1458.

2-(4-(4-fluorobenzoyl)piperazin-1-yl)acetohydrazide (46g)

Synthesized according to General Procedure C: 51g (3.73 g, 12.7 mmol,1.0 equiv.), anhydrous hydrazine (1.6 mL, 50.8 mmol, 4.0 equiv.), EtOH(25 mL, 0.5 M). 46g (2.28 g, 64.1%) was obtained as a white solid afterextraction without further purification. ¹H-NMR (500 MHz, CDCl₃) δ¹H-NMR (500 MHz, CDCl₃) δ 8.09 (br s, 1H), 7.37-7.33 (m, 2H), 7.06-7.02(m, 2H), 3.85 (br s, 2H), 3.70 (br s, 2H), 3.42 (br s, 2H), 3.06 (s,2H), 2.48 (br s, 4H). ¹³C-NMR (125 MHz, CDCl₃) δ 169.8, 169.5, 163.5 (d,J_(C-F)=249.1 Hz), 131.5 (d, J_(C-F)=2.8 Hz), 129.4 (d, J_(C-F)=9.1 Hz),115.7 (d, J_(C-F)=22.0 Hz), 60.6, 53.5 (br), 47.7 (br), 42.3 (br).¹⁹F-NMR (470 MHz, CDCl₃) δ −113.1. HRMS (ESI): 281.1409 (M+1); calcd.for C₁₃H_(1s)FN₄O₂: 281.1414.

Ethyl 2-(4-(4-(trifluoromethyl)benzoyl)piperazin-1-yl)acetate (51i)

Synthesized according to General Procedure B: 50 (2.58 g, 15.0 mmol, 1.0equiv.), anhydrous tetrahydrofuran (30 mL, 0.5 M), freshly distilledEt₃N (4.2 mL, 30.0 mmol, 2.0 equiv.), 4-(trifluoroemthyl)benzoylchloride (54i, 2.2 mL, 15.0 mmol, 1.0 equiv.). Purification bysilica-gel column chromatography (50-100% EtOAc/hexanes) afforded 51i(4.01 g, 77.5%) as a yellow oil. ¹H-NMR (500 MHz, CDCl₃) δ 7.65 (d, 2H,J=8.0 Hz), 7.49 (d, 2H, J=8.0 Hz), 4.16 (q, 2H, J=7.0 Hz), 3.82 (br s,2H), 3.41 (br s, 2H), 3.24 (s, 2H), 2.68 (br s, 2H), 2.54 (br s, 2H),1.24 (t, 3H, J=7.0 Hz). ¹³C-NMR (125 MHz, CDCl₃) δ170.1, 168.9, 139.4131.8 (q, J_(C-F)=32.6 Hz), 127.6, 125.7 (q, J_(C-F)=3.8 Hz), 123.8 (q,J_(C-F)=271.0 Hz), 60.9, 59.1, 53.0 (br), 52.5 (br), 47.6 (br), 42.2(br), 14.3. ¹⁹F-NMR (470 MHz, CDCl₃) δ −66.0. HRMS (ESI): 345.1430(M+1); calcd. for C₁₆H₂₀F₃N₂O₃: 345.1426.

2-(4-(4-(trifluoromethyl)benzoyl)piperazin-1-yl)acetohydrazide (46i)

Synthesized according to General Procedure C: 51i (4.00 g, 11.6 mmol,1.0 equiv.), anhydrous hydrazine (1.5 mL, 46.4 mmol, 4.0 equiv.), EtOH(25 mL, 0.5 M). 46i (2.35 g, 61.4%) was obtained as a white solid afterextraction without further purification. ¹H-NMR (500 MHz, CDCl₃) δ 8.09(br s, 1H), 7.62 (d, 2H, J=8.0 Hz), 7.45 (d, 2H, J=8.0 Hz), 3.88 (br s,2H) 3.75 (br s, 2H), 3.35 (br s, 2H), 3.07 (s, 2H), 2.56 (br s, 2H),2.42 (br s, 2H). ¹³C-NMR (125 MHz, CDCl₃) δ169.8, 168.9, 139.1 131.8 (q,J_(C-F)=32.1 Hz), 127.5, 125.7 (q, J_(C-F)=3.6 Hz), 123.7 (q,J_(C-F)=271.1 Hz), 60.5, 53.7 (br), 53.2 (br), 47.6 (br), 42.2 (br).¹⁹F-NMR (470 MHz, CDCl₃) δ

−66.0. HRMS (ESI): 331.1374 (M+1); calcd. for C₁₄H₁₈F₃N₄O₂: 331.1382.

3-propylsalicylaldehyde (47b)

To a round-bottom flask were added aldehyde 47a (1.62 g, 10.0 mmol, 1.0equiv.), 5% Pd/C (324 mg, 20 wt % of 47a), diphenyl sulfide (17 μL, 0.10mmol, 0.010 equiv.), and EtOAc (40 mL, 0.25 M). The reaction mixture wasstirred overnight at room temperature under an atmosphere of H₂ (balloonpressure). The reaction mixture was filtered through Celite and washedthoroughly with EtOAc. The filtrate was concentrated to afford aldehyde47b (1.50 g, 91.7%) as a yellow oil. ¹H-NMR (500 MHz, CDCl₃) δ 11.27 (s,1H), 9.88 (s, 1H), 7.41-7.38 (m, 2H), 6.95 (t, 1H, J=7.5 Hz), 2.64 (t,2H, J=7.5 Hz), 1.65 (sext, 2H, J=7.5 Hz), 0.96 (t, 3H, J=7.5 Hz).¹³C-NMR (125 MHz, CDCl₃) δ 197.0, 160.0, 137.4, 131.7, 131.4, 120.4,119.6, 31.3, 22.7, 14.1. HRMS (EI): 164.08383 (M⁺); calcd. for C₁₀H₁₂O₂:164.08373.

2-(allyloxy)-5-fluorobenzaldehyde (53b)

To a round bottom flask were added 5-fluorosalicylaldehyde (47c, 4.0 g,28.5 mmol, 1.0 equiv.), potassium carbonate (4.92 g, 35.6 mmol, 1.25equiv.), and DMF (20 mL). Allyl bromide (3.7 mL, 42.8 mmol, 1.5 equiv.)was added slowly to the mixture. The reaction mixture was stirredovernight at room temperature. The reaction mixture was diluted withwater (50 mL) and extracted with ethyl acetate (3×50 mL). The combinedorganic layers were washed with water (2×25 mL), 0.1M KOH (2×25 mL),water (2×25 mL), and brine (2×25 mL), dried over MgSO₄, filtered, andconcentrated to yield 53b (4.64 g, 90.2%) as a pale yellow liquid.¹H-NMR (500 MHz, CDCl₃) δ 10.47 (d, 1H, J=3.0 Hz), 7.50 (dd, 1H, J=3.0,8.0 Hz), 7.23 (ddd, 1H, J=3.0, 7.5, 11.0 Hz), 6.95 (dd, 1H, J=4.0, 9.0Hz), 6.06 (tdd, 1H, J=5.0, 10.5, 17.5 Hz), 5.44 (qd, 1H, J=1.5, 17.0Hz), 5.34 (ddd, 1H, J=1.5, 2.5, 10.5 Hz), 4.64 (td, 2H, J=1.5, 5.0 Hz).¹³C-NMR (125 MHz, CDCl₃) δ 188.8, 157.4 (d, J_(C-F)=1.9 Hz), 157.2 (d,J_(C-F)=240.5 Hz), 132.4, 126.1 (d, J_(C-F)=5.9 Hz), 122.6 (d,J_(C-F)=23.8 Hz), 118.5, 114.8 (d, J_(C-F)=7.1 Hz), 114.2 (d,J_(C-F)=23.1 Hz), 70.1. ¹⁹F-NMR (470 MHz, CDCl₃) δ −125.5. HRMS (EI):180.05789 (M⁺); calcd. for C₁₀H₉FO₂: 180.05866.

3-allyl-5-fluorosalicylaldehyde (47d)

53b (4.64 g, 25.8 mmol) was heated neat overnight at 200° C. The crudeproduct was purified by silica gel column chromatography (hexanes) toyield 47d (2.24 g, 48.3%) as a bright yellow oil. ¹H-NMR (500 MHz,CDCl₃) δ 11.10 (s, 1H), 9.83 (s, 1H), 7.17 (dd, 1H, J=3.0, 9.0 Hz), 7.11(dd, 1H, J=3.0, 7.5 Hz) 5.96 (tdd, 1H, J=6.5, 10.0, 17.0 Hz), 5.16-5.14(m, 1H), 5.12 (qd, 1H, J=1.5, 11.0 Hz), 3.42 (d, 2H, J=6.5 Hz). ¹³C-NMR(125 MHz, CDCl₃) δ 195.9 (d, J_(C-F)=2.5 Hz), 156.0 (d, J_(C-F)=1.0 Hz),155.7 (d, J_(C-F)=238.8 Hz), 135.1, 131.6 (d, J_(C-F)=6.4 Hz), 124.8 (d,J_(C-F)=23.6 Hz), 119.8 (d, J_(C-F)=6.4 Hz), 117.3, 116.0 (d,J_(C-F)=22.3 Hz), 33.2. ¹⁹F-NMR (470 MHz, CDCl₃) δ −126.9. HRMS (EI):180.05761 (M⁺); calcd. for C₁₀H₉FO₂: 180.05866.

5-fluoro-2-hydroxy-3-propylbenzaldehyde (47e)

To a round-bottom flask were added aldehyde 47d (1.10 g, 6.11 mmol, 1.0equiv.), 5% Pd/C (220 mg, 20 wt % of 47d), diphenyl sulfide (10 μL,0.061 mmol, 0.010 equiv.), and EtOAc (25 mL, 0.25 M). The reactionmixture was stirred overnight at room temperature under an atmosphere ofH₂ (balloon pressure). The reaction mixture was filtered through Celiteand washed thoroughly with EtOAc. The filtrate was concentrated toafford aldehyde 47e (991 mg, 89.3%) as a yellow oil. ¹H-NMR (500 MHz,CDCl₃) δ 11.06 (br s, 1H), 9.80 (s, 1H), 7.12 (dd, 1H, J=3.0, 9.0 Hz),7.06 (dd, 1H, J=3.0, 7.5 Hz), 2.62 (t, 2H, J=7.5 Hz), 1.63 (sext, 2H,J=7.5 Hz), 0.96 (t, 3H, J=7.5 Hz). ¹³C-NMR (125 MHz, CDCl₃) δ 195.9 (d,J_(C-F)=2.5 Hz), 156.3 (d, J_(C-F)=1.0 Hz), 155.5 (d, J_(C-F)=238.1 Hz),133.9 (d, J_(C-F)=6.3 Hz), 124.7 (d, J_(C-F)=23.1 Hz), 119.6 (d,J_(C-F)=6.5 Hz), 115.4 (d, J_(C-F)=22.4 Hz), 31.2, 22.4, 14.0. ¹⁹F-NMR(470 MHz, CDCl₃) δ −127.3. HRMS (EI): 182.07392 (M⁺); calcd. forC₁₀H₁₁FO₂: 182.07431.

General Procedure D: Synthesis of PAC-1 Analogues

To a 16×150 mm test tube were added hydrazide (1.0 equiv.), aldehyde(1.0 equiv.), EtOH or 2:1 MeOH:MeCN (0.15 M), and 1.2 M HCl (7 mol %).The reaction mixture was shaken overnight at reflux on a Büchi Syncoreparallel synthesizer. The reaction mixture was cooled to roomtemperature, concentrated, and purified by silica gel columnchromatography or recrystallization to yield pure PAC-1 analogue.

N′-(3-allyl-2-hydroxybenzylidene)-2-(4-benzoylpiperazin-1-yl)acetohydrazide(3)

Synthesized according to General Procedure D, but in a round-bottomflask: 46c (262 mg, 1.0 mmol, 1.0 equiv.), 47a (162 mg, 1.0 mmol, 1.0equiv.), 1.2 M HCl (58 μL, 0.070 mmol, 0.070 equiv.), EtOH (7 mL, 0.15M). Purification by silica gel column chromatography (gradient, 0-10%MeOH/EtOAc) yielded 3 (284 mg, 69.8%) as a white solid. ¹H-NMR (500 MHz,CDCl₃) δ 11.19 (s, 1H), 9.94 (br s, 1H), 8.45 (s, 1H), 7.46-7.41 (m,5H), 7.20 (d, 1H, J=6.5 Hz), 7.08 (dd, 1H, J=1.5, 7.5 Hz), 6.85 (t, 1H,J=7.0 Hz), 6.03 (tdd, 1H, J=6.5, 10.0, 16.5 Hz), 5.10-5.05 (m, 2H), 3.88(br s, 2H), 3.58 (s, 2H), 3.52 (br s, 2H), 3.45 (d, 2H, J=6.5 Hz), 3.25(s, 2H), 2.68 (br s, 2H), 2.61 (br s, 2H). ¹³C-NMR (125 MHz, CDCl₃) δ170.6, 165.4, 156.4, 151.6, 136.5, 135.4, 132.5, 130.2, 129.3, 128.8,128.3, 127.1, 119.2, 116.9, 115.8, 61.0, 53.7, 53.1, 47.6, 42.1, 33.9.HRMS (ESI): 407.2077 (M+1); calcd. for C₂₃H₂₇N₄O₃: 407.2083.

N′-(3-allyl-2-hydroxybenzylidene)-2-(4-(4-cyanobenzyl)piperazin-1-yl)acetohydrazide(4)

Synthesized according to General Procedure D: 46d (273 mg, 1.0 mmol, 1.0equiv.), 47a (162 mg, 1.0 mmol, 1.0 equiv.), 1.2 M HCl (58 μL, 0.070mmol, 0.070 equiv.), EtOH (7 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-15% MeOH/EtOAc) yielded 4 (367 mg,87.7%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.25 (br s, 1H),9.99 (br s, 1H), 8.40 (s, 1H), 7.61 (d, 2H, J=8.0 Hz), 7.44 (d, 2H,J=8.0 Hz), 7.18 (dd, 1H, J=1.5, 7.5 Hz), 7.07 (dd, 1H, J=1.5, 7.5 Hz),6.84 (t, 1H, J=7.5 Hz), 6.02 (tdd, 1H, J=6.5, 10.0, 16.5 Hz), 5.11-5.04(m, 2H), 3.58 (s, 2H), 3.44 (d, 2H, J=7.0 Hz), 3.19 (s, 2H), 2.63 (br s,4H), 2.53 (br s, 4H). ¹³C-NMR (125 MHz, CDCl₃) δ 165.9, 156.5, 151.5,144.0, 136.6, 132.5, 132.4, 129.6, 129.3, 128.4, 119.2, 119.1, 117.0,115.8, 111.2, 62.4, 61.1, 53.8, 53.2, 34.0. HRMS (ESI): 418.2242 (M+1);calcd. for C₂₄H₂₈N₅O₂: 418.2243.

N′-(3-allyl-2-hydroxybenzylidene)-2-(4-(4-cyanobenzoyl)piperazin-1-yl)acetohydrazide(5)

Synthesized according to General Procedure D: 46e (287 mg, 1.0 mmol, 1.0equiv.), 47a (162 mg, 1.0 mmol, 1.0 equiv.), 1.2 M HCl (58 μL, 0.070mmol, 0.070 equiv.), EtOH (7 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-10% MeOH/EtOAc) yielded 5 (378 mg,87.6%) as a light yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.23 (br s,1H), 9.98 (br s, 1H), 8.32 (s, 1H), 7.69 (d, 2H, J=8.5 Hz), 7.48 (d, 2H,J=8.0 Hz), 7.17 (d, 1H, J=7.0 Hz), 6.99 (dd, 1H, J=1.5, 8.0 Hz), 6.81(t, 1H, J=7.5 Hz), 5.98 (tdd, 1H, J=6.5, 10.0, 17.0), 5.08-5.02 (m, 2H),3.85 (br s, 2H), 3.42-3.39 (m, 4H), 3.23 (s, 2H), 2.68 (br s, 2H), 2.86(br s, 4H). ¹³C-NMR (125 MHz, CDCl₃) δ 168.4, 165.3, 156.4, 151.7,139.7, 136.5, 132.6, 132.6, 129.4, 128.2, 127.9, 119.3, 118.1, 116.8,115.9, 113.8, 60.9, 53.7 (br), 53.3 (br), 47.5 (br), 42.1 (br), 33.9.HRMS (ESI): 432.2034 (M+1); calcd. for C₂₄H₂₆N₅O₃: 432.2036.

N′-(3-allyl-2-hydroxybenzylidene)-2-(4-(4-fluorobenzyl)piperazin-1-yl)acetohydrazide(6)

Synthesized according to General Procedure D: H11 (133 mg, 0.50 mmol,1.0 equiv.), 47a (81 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-10% MeOH/EtOAc) followed byprecipitation from Et₂O yielded 6 (182 mg, 89.0%) as a white solid.¹H-NMR (500 MHz, CDCl₃) δ 11.26 (br s, 1H), 10.02 (br s, 1H), 8.41 (s,1H), 7.29-7.26 (m, 2H), 7.19 (dd, 1H, J=1.5, 7.5 Hz), 7.08 (dd, 1H,J=1.5, 8.0 Hz), 7.02-6.99 (m, 2H), 6.85 (t, 1H, J=7.5 Hz), 6.03 (tdd,1H, J=6.5, 10.0, 16.5 Hz), 5.11-5.04 (m, 2H), 3.50 (s, 2H), 3.45 (d, 2H,J=6.5 Hz), 3.19 (s, 2H), 2.62 (br s, 4H), 2.51 (br s, 4H). ¹³C-NMR (125MHz, CDCl₃) δ 166.0, 162.2 (d, J_(C-F)=243.9 Hz), 156.6, 151.5, 136.7,133.7 (d, J_(C-F)=3.1 Hz), 132.5, 130.7 (d, J_(C-F)=7.8 Hz), 129.3,128.4, 119.2, 117.0, 115.8, 115.3 (d, J_(C-F)=21.0 Hz), 62.2, 61.2,53.9, 53.1, 34.0. ¹⁹F-NMR (470 MHz, CDCl₃) δ −118.8. HRMS (ESI):411.2203 (M+1); calcd. for C₂₃H₂₈FN₄O₂: 411.2196.

N′-(3-allyl-2-hydroxybenzylidene)-2-(4-(4-fluorobenzoyl)piperazin-1-yl)acetohydrazide(7)

Synthesized according to General Procedure D: H12 (140 mg, 0.50 mmol,1.0 equiv.), 47a (81 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-10% MeOH/EtOAc) yielded 7 (171 mg,80.5%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.19 (br s, 1H),9.91 (br s, 1H), 8.43 (s, 1H), 7.43-7.40 (m, 2H), 7.19 (dd, 1H, J=1.0,7.5 Hz), 7.12-7.09 (m, 2H), 7.06 (dd, 1H, J=1.5, 8.0 Hz), 6.85 (t, 1H,J=7.5 Hz), 6.02 (tdd, 1H, J=6.5, 10.0, 16.5 Hz), 5.10-5.04 (m, 2H), 3.69(br s, 4H), 3.44 (d, 2H, J=6.5 Hz), 3.24 (s, 2H), 2.64 (br s, 4H).¹³C-NMR (125 MHz, CDCl₃) δ 169.6, 165.4, 163.6 (d, J_(C-F)=249.1 Hz),156.4, 151.6, 136.5, 132.5, 131.4 (d, J_(C-F)=3.4 Hz), 129.5 (d,J_(C-F)=8.4 Hz), 129.3, 128.2, 119.2, 116.8, 115.8, 115.8 (d,J_(C-F)=21.5 Hz), 60.9, 53.6 (br), 47.7 (br), 42.3 (br), 33.9. ¹⁹F-NMR(470 MHz, CDCl₃) δ −112.8. HRMS (ESI): 425.1989 (M+1); calcd. forC₂₃H₂₆FN₄O₃: 425.1989.

N′-(3-allyl-2-hydroxybenzylidene)-2-(4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)acetohydrazide(8)

Synthesized according to General Procedure D: H13 (158 mg, 0.50 mmol,1.0 equiv.), 47a (81 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-15% MeOH/EtOAc) yielded 8 (125 mg,54.4%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.32 (br s, 1H),10.11 (br s, 1H), 8.33 (s, 1H), 7.56 (d, 2H, J=8.5 Hz), 7.43 (d, 2H,J=8.0 Hz), 7.17 (dd, 1H, J=1.5, 7.5 Hz), 7.04 (dd, 1H, J=1.5, 8.0 Hz),6.83 (t, 1H, J=7.5 Hz), 6.02 (tdd, 1H, J=6.5, 10.0, 16.5 Hz), 5.10-5.04(m, 2H), 3.57 (s, 2H), 3.44 (d, 2H, J=7.0 Hz), 3.19 (s, 2H), 2.63 (br s,4H), 2.53 (br s, 4H). ¹³C-NMR (125 MHz, CDCl₃) δ 166.0, 156.4, 151.2,142.4, 136.6, 132.4, 129.5 (q, J_(C-F)=32.0 Hz), 129.3, 129.3, 128.3,125.3 (q, J_(C-F)=3.8 Hz), 123.9 (q, J_(C-F)=270.6 Hz), 119.2, 117.0,115.8, 62.3, 61.0, 53.7, 53.1, 34.0. ¹⁹F-NMR (470 MHz, CDCl₃) δ −65.4.HRMS (ESI): 461.2160 (M+1); calcd. for C₂₄H₂₈F₃N₄O₂: 461.2164.

N′-(3-allyl-2-hydroxybenzylidene)-2-(4-(4-(trifluoromethyl)benzoyl)piperazin-1-yl)acetohydrazide(9)

Synthesized according to General Procedure D: H14 (165 mg, 0.50 mmol,1.0 equiv.), 47a (81 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-15% MeOH/EtOAc) yielded 9 (211 mg,89.1%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.28 (br s, 1H),10.13 (br s, 1H), 8.27 (s, 1H), 7.65 (d, 2H, J=8.0 Hz), 7.48 (d, 2H,J=8.0 Hz), 7.15 (d, 1H, J=8.0 Hz), 6.94 (d, 2H, J=7.0 Hz), 6.79 (t, 1H,J=7.5 Hz), 5.97 (tdd, 1H, J=6.5, 10.0, 17.0 Hz), 5.05-5.00 (m, 2H), 3.86(br s, 2H), 3.43 (br s, 2H), 3.39 (d, 2H, J=6.5 Hz), 3.21 (s, 2H), 2.66(br s, 2H), 2.58 (br s, 2H). ¹³C-NMR (125 MHz, CDCl₃) δ 169.0, 165.4,156.3, 151.5, 139.0, 136.4, 132.5, 131.9 (q, J_(C-F)=32.6 Hz), 129.3,128.2, 127.5, 125.8 (q, J_(C-F)=3.5 Hz), 123.7 (q, J_(C-F)=271.1 Hz),119.3, 116.8, 115.8, 60.8, 53.5 (br), 47.5 (br), 42.0 (br), 33.8.¹⁹F-NMR (470 MHz, CDCl₃) δ −66.0. HRMS (ESI): 475.1964 (M+1); calcd. forC₂₄H₂₆F₃N₄O₃: 475.1957.

2-(4-benzylpiperazin-1-yl)-N′-(2-hydroxy-3-propylbenzylidene)acetohydrazide(10)

Synthesized according to General Procedure D, but in a round-bottomflask: 46a (248 mg, 1.0 mmol, 1.0 equiv.), 47b (164 mg, 1.0 mmol, 1.0equiv.), 1.2 M HCl (58 μL, 0.070 mmol, 0.070 equiv.), EtOH (7 mL, 0.15M). Purification by silica gel column chromatography (gradient, 0-20%MeOH/EtOAc) yielded 10 (345 mg, 87.3%) as an off-white solid. ¹H-NMR(500 MHz, CDCl₃) δ 11.30 (s, 1H), 10.12 (br s, 1H), 8.31 (s, 1H),7.35-7.30 (m, 4H), 7.30-7.25 (m, 1H), 7.17 (d, 1H, J=7.5 Hz), 7.03 (d,1H, J=7.5 Hz), 6.82 (t, 1H, J=7.5 Hz), 3.54 (s, 2H), 3.19 (s, 2H), 2.67(t, 2H, J=7.5 Hz), 2.62 (br s, 4H), 2.54 (br s, 4H), 1.67 (sext, 2H,J=7.5 Hz), 0.97 (t, 3H, J=7.5 Hz). ¹³C-NMR (125 MHz, CDCl₃) δ 165.9,156.7, 151.2, 137.9, 132.5, 130.7, 129.2, 128.8, 128.4, 127.3, 118.9,116.8, 62.9, 61.0, 53.7, 53.0, 32.0, 22.7, 14.2. HRMS (ESI): 395.2436(M+1); calcd. for C₂₃H₃₁N₄O₂: 395.2447.

4-((4-(2-(2-(2-hydroxy-3-propylbenzylidene)hydrazinyl)-2-oxoethyl)piperazin-1-yl)methyl)benzenesulfonamide(11)

Synthesized according to General Procedure D: H2 (164 mg, 0.50 mmol, 1.0equiv.), 47b (82 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), 2:1 MeOH:MeCN (3 mL, 0.15 M). Purification bysilica gel column chromatography (gradient, 0-20% MeOH/EtOAc) yielded 11(211 mg, 89.0%) as a white solid. ¹H-NMR (500 MHz, (CD₃)₂CO) δ 11.78 (s,1H), 10.76 (br s, 1H), 8.48 (s, 1H), 7.84 (d, 2H, J=8.5 Hz), 7.51 (d,2H, J=8.5 Hz), 7.17 (d, 1H, J=7.0 Hz), 7.14 (dd, 1H, J=1.5, 8.0 Hz),6.82 (t, 1H, J=7.5 Hz), 6.54 (br s, 2H), 3.59 (s, 2H), 3.17 (s, 2H),2.64-2.59 (m, 6H), 2.52 (br s, 4H), 1.63 (sext, 2H, J=7.5 Hz), 0.93 (t,3H, J=7.5 Hz). ¹³C-NMR (125 MHz, (CD₃)₂CO) δ 166.3, 157.3, 150.9, 144.0,143.8, 132.7, 130.8, 129.9, 129.6, 126.9, 119.6, 118.3, 62.6, 61.7,54.3, 53.6, 32.5, 23.4, 14.2. HRMS (ESI): 474.2175 (M+1); calcd. forC₂₃H₃₂N₅O₄S: 474.2175.

2-(4-benzoylpiperazin-1-yl)-N′-(2-hydroxy-3-propylbenzylidene)acetohydrazide(12)

Synthesized according to General Procedure H3 (131 mg, 0.50 mmol, 1.0equiv.), 47b (82 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 50-100% EtOAc/hexanes, then 5%MeOH/EtOAc) yielded 12 (174 mg, 85.5%) as a white solid. ¹H-NMR (500MHz, CDCl₃) δ 11.29 (s, 1H), 10.29 (br s, 1H), 8.23 (s, 1H), 7.41-7.34(m, 5H), 7.13 (dd, 1H, J=1.5, 7.5 Hz), 6.90 (dd, 1H, J=1.5, 7.5 Hz),6.76 (t, 1H, J=7.5 Hz), 3.80 (br s, 2H), 3.47 (br s, 2H), 3.18 (s, 2H),2.71-2.52 (m, 6H, Ar—CH₂—CH₂), 1.61 (sext, 2H, J=7.5 Hz), 0.91 (t, 3H,J=7.5 Hz). ¹³C-NMR (125 MHz, CDCl₃) δ 170.5, 165.5, 156.6, 151.5, 135.3,132.5, 130.6, 130.1, 128.8, 128.7, 127.0, 118.9, 116.7, 60.8, 53.6 (br),53.0 (br), 47.5 (br), 42.0 (br), 31.9, 22.7, 14.1. HRMS (ESI): 409.2238(M+1); calcd. for C₂₃H₂₉N₄O₃: 409.2240.

2-(4-(4-cyanobenzyl)piperazin-1-yl)-N′-(2-hydroxy-3-propylbenzylidene)acetohydrazide(13)

Synthesized according to General Procedure D: H9 (273 mg, 1.0 mmol, 1.0equiv.), 47b (164 mg, 1.0 mmol, 1.0 equiv.), 1.2 M HCl (58 μL, 0.070mmol, 0.070 equiv.), EtOH (7 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-15% MeOH/EtOAc) yielded 13 (373 mg,88.8%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.19 (br s, 1H),9.99 (br s, 1H), 8.37 (s, 1H), 7.60 (d, 2H, J=8.0 Hz), 7.44 (d, 2H,J=7.5 Hz), 7.16 (dd, 1H, J=1.5, 7.5 Hz), 7.04 (dd, 1H, J=1.5, 7.5 Hz),6.82 (t, 1H, J=7.5 Hz), 3.58 (s, 2H), 3.19 (s, 2H), 2.66-2.63 (m, 6H),2.52 (br s, 4H), 1.64 (sext, 2H, J=7.5 Hz), 0.95 (t, 3H, J=7.5 Hz).¹³C-NMR (125 MHz, CDCl₃) δ 165.8, 156.8, 151.6, 144.0, 132.7, 132.3,130.8, 129.6, 128.9, 119.1, 119.0, 116.8, 111.2, 62.4, 61.1, 53.8, 53.2,32.0, 22.8, 14.2. HRMS (ESI): 420.2396 (M+1); calcd. for C₂₄H₃₀N₅O₂:420.2400.

2-(4-(4-cyanobenzoyl)piperazin-1-yl)-N′-(2-hydroxy-3-propylbenzylidene)acetohydrazide(14)

Synthesized according to General Procedure D: H10 (287 mg, 1.0 mmol, 1.0equiv.), 47b (164 mg, 1.0 mmol, 1.0 equiv.), 1.2 M HCl (58 μL, 0.070mmol, 0.070 equiv.), EtOH (7 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-15% MeOH/EtOAc) yielded 14 (377 mg,86.9%) as a light yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.15 (br s,1H), 9.92 (br s, 1H), 8.32 (s, 1H), 7.71 (d, 2H, J=8.0 Hz), 7.49 (d, 2H,J=7.5 Hz), 7.16 (d, 1H, J=7.0 Hz), 6.98 (dd, 1H, J=1.5, 7.5 Hz), 6.80(t, 1H, J=7.5 Hz), 3.86 (br s, 2H), 3.44 (br s, 2H), 3.24 (s, 2H), 2.70(br s, 2H), 2.64-2.57 (m, 4H), 1.62 (sext, 2H, J=7.5 Hz), 0.93 (t, 3H,J=7.5 Hz). ¹³C-NMR (125 MHz, CDCl₃) δ 168.5, 165.2, 156.7, 152.0, 139.8,132.9, 132.7, 130.8, 129.0, 127.9, 119.1, 118.1, 116.7, 113.9, 61.0,53.5 (br), 47.5 (br), 42.1 (br), 32.0, 22.8, 14.2. HRMS (ESI): 434.2188(M+1); calcd. for C₂₄H₂₈N₅O₃: 434.2192.

2-(4-(4-fluorobenzyl)piperazin-1-yl)-N′-(2-hydroxy-3-propylbenzylidene)acetohydrazide(15)

Synthesized according to General Procedure D: H11 (133 mg, 0.50 mmol,1.0 equiv.), 47b (82 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) followed byprecipitation from Et₂O yielded 15 (137 mg, 66.4%) as a white solid.¹H-NMR (500 MHz, CDCl₃) δ 11.26 (br s, 1H), 10.09 (br s, 1H), 8.31 (s,1H), 7.26 (dd, 2H, J=6.0, 8.0 Hz), 7.16 (dd, 1H, J=1.5, 6.5 Hz),7.02-6.97 (m, 3H), 6.80 (t, 1H, J=7.5 Hz), 3.48 (s, 2H), 3.18 (s, 2H),2.65 (t, 2H, J=7.5 Hz), 2.61 (br s, 4H), 2.50 (br s, 4H), 1.65 (sext,2H, J=7.5 Hz), 0.95 (t, 3H, J=7.5 Hz). ¹³C-NMR (125 MHz, CDCl₃) δ 165.9,162.1 (d, J_(C-F)=243.6 Hz), 156.7, 151.3, 133.7 (d, J_(C-F)=3.0 Hz),132.5, 130.7, 130.6 (d, J_(C-F)=7.8 Hz), 128.9, 118.9, 116.8, 115.2 (d,J_(C-F)=21.0 Hz), 62.1, 61.0, 53.8, 53.0, 32.0, 22.8, 14.2. ¹⁹F-NMR (470MHz, CDCl₃) δ −118.8. HRMS (ESI): 413.2361 (M+1); calcd. forC₂₃H₃₀FN₄O₂: 413.2353.

2-(4-(4-fluorobenzoyl)piperazin-1-yl)-N′-(2-hydroxy-3-propylbenzylidene)acetohydrazide(16)

Synthesized according to General Procedure D: H12 (140 mg, 0.50 mmol,1.0 equiv.), 47b (82 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-15% MeOH/EtOAc) yielded 16 (133 mg,62.4%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.24 (br s, 1H),10.17 (br s, 1H), 8.25 (s, 1H), 7.37 (dd, 2H, J=5.5, 8.5 Hz), 7.13 (dd,1H, J=1.5, 7.5 Hz), 7.06 (t, 2H, J=8.5 Hz), 6.91 (dd, 1H, J=1.5, 7.5Hz), 6.77 (t, 1H, J=7.5 Hz), 3.83 (br s, 2H), 3.49 (br s, 2H), 3.20 (s,2H), 2.62-2.58 (m, 6H), 1.60 (sext, 2H, J=7.5 Hz), 0.91 (t, 3H, J=7.5Hz). ¹³C-NMR (125 MHz, CDCl₃) δ 169.6, 165.4, 163.6 (d, J_(C-F)=249.3Hz), 156.6, 151.7, 132.6, 131.4 (d, J_(C-F)=3.4 Hz), 129.5 (d,J_(C-F)=8.5 Hz), 128.9, 119.0, 116.7, 115.8 (d, J_(C-F)=21.8 Hz), 60.9,53.5 (br), 47.7 (br), 42.2 (br), 31.9, 22.7, 14.1. ¹⁹F-NMR (470 MHz,CDCl₃) δ −112.8. HRMS (ESI): 427.2141 (M+1); calcd. for C₂₃H₂₈FN₄O₃:427.2145.

N′-(2-hydroxy-3-propylbenzylidene)-2-(4-(4-(trifluoromethybbenzyppiperazin-1-yl)acetohydrazide(17)

Synthesized according to General Procedure D: H13 (158 mg, 0.50 mmol,1.0 equiv.), 47b (82 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-15% MeOH/EtOAc) yielded 17 (93.9 mg,40.6%) as a yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.23 (br s, 1H),10.05 (br s, 1H), 8.33 (s, 1H), 7.57 (d, 2H, J=8.0 Hz), 7.44 (d, 2H,J=8.0 Hz), 7.16 (dd, 1H, J=1.5, 7.5 Hz), 7.02 (dd, 2H, J=1.5, 7.5 Hz),6.81 (t, 1H, J=7.5 Hz), 3.58 (s, 2H), 3.19 (s, 2H), 2.67-2.62 (m, 6H),2.53 (br s, 4H), 1.65 (sext, 2H, J=7.5 Hz), 0.95 (t, 3H, J=7.5 Hz).¹³C-NMR (125 MHz, CDCl₃) δ 165.9, 156.8, 151.5, 142.4, 132.6, 130.8,129.6 (q, J_(C-F)=32.0 Hz), 129.3, 128.9, 125.4 (q, J_(C-F)=3.6 Hz),124.4 (q, J_(C-F)=270.5 Hz), 119.0, 116.9, 62.4, 61.1, 53.8, 53.2, 32.0,22.8, 14.2. ¹⁹F-NMR (470 MHz, CDCl₃) δ −65.5. HRMS (ESI): 463.2321(M+1); calcd. for C₂₄H₃₀F₃N₄O₂: 463.2321.

N′-(2-hydroxy-3-propylbenzylidene)-2-(4-(4-(trifluoromethyl)benzoyl)piperazin-1-yl)acetohydrazide(18)

Synthesized according to General Procedure D: H14 (165 mg, 0.50 mmol,1.0 equiv.), 47b (82 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-15% MeOH/EtOAc) yielded 18 (216 mg,90.8%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.24 (br s, 1H),10.14 (br s, 1H), 8.23 (s, 1H), 7.64 (d, 2H, J=8.0 Hz), 7.47 (d, 2H,J=8.0 Hz), 7.13 (d, 1H, J=8.0 Hz), 6.89 (d, 1H, J=7.5 Hz), 6.76 (t, 1H,J=7.5 Hz), 3.85 (br s, 2H), 3.43 (br s, 2H), 3.21 (s, 2H), 2.73-2.58 (m,6H), 1.60 (sext, 2H, J=7.5 Hz), 0.90 (t, 3H, J=7.5 Hz). ¹³C-NMR (125MHz, CDCl₃) δ 169.0, 165.4, 156.5, 151.6, 139.0, 132.7, 131.9 (q,J_(C-F)=32.5 Hz), 130.6, 128.8, 127.5, 125.8 (q, J_(C-F)=3.5 Hz), 123.7(q, J_(C-F)=271.3 Hz), 119.0, 116.7, 60.8, 53.5 (br), 47.5 (br), 42.0(br), 31.9, 22.7, 14.1. ¹⁹F-NMR (470 MHz, CDCl₃) δ −66.0. HRMS (ESI):477.2108 (M+1); calcd. for C₂₄H₂₈F₃N₄O₃: 477.2114.

2-(4-benzylpiperazin-1-yl)-N′-(5-fluoro-2-hydroxybenzylidene)acetohydrazide(19)

Synthesized according to General Procedure D: H1 (124 mg, 0.50 mmol, 1.0equiv.), 47c (70 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) yielded 19 (173 mg,93.7%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 10.81 (br s, 1H),10.13 (br s, 1H), 8.39 (s, 1H), 7.33-7.30 (m, 4H), 7.28-7.25 (m, 1H),7.00 (dt, 1H, J=3.0, 9.0 Hz), 6.94-6.89 (m, 2H), 3.55 (s, 2H), 3.19 (s,2H), 2.63 (br s, 4H), 2.53 (br s, 4H). ¹³C-NMR (125 MHz, CDCl₃) δ 166.3,155.9 (d, J_(C-F)=235.8 Hz), 154.8, 150.0, 137.9, 129.3, 128.5, 127.4,118.9 (d, J_(C-F)=23.1 Hz), 118.4 (d, J_(C-F)=7.6 Hz), 117.6 (d,J_(C-F)=7.5 Hz), 116.1 (d, J_(C-F)=23.8 Hz), 63.0, 61.1, 53.9, 53.1.¹⁹F-NMR (470 MHz, CDCl₃) δ −128.5. HRMS (ESI): 371.1877 (M+1); calcd.for C₂₀H₂₄FN₄O₂: 371.1883.

4-((4-(2-(2-(5-fluoro-2-hydroxybenzylidene)hydrazinyl)-2-oxoethyl)piperazin-1-yl)methyl)benzenesulfonamide(20)

Synthesized according to General Procedure D: H2 (164 mg, 0.50 mmol, 1.0equiv.), 47c (70 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), 2:1 MeOH:MeCN (3 mL, 0.15 M). Purification bysilica gel column chromatography (gradient, 0-20% MeOH/EtOAc) yielded 20(172 mg, 82.1%) as a yellow solid. ¹H-NMR (500 MHz, (CD₃)₂CO) δ 11.33(br s, 1H), 10.95 (br s, 1H), 8.49 (s, 1H), 7.85 (d, 2H, J=8.0 Hz), 7.49(d, 2H, J=8.0 Hz), 7.12 (dd, 1H, J=3.0, 9.0 Hz), 7.07 (dt, 1H, J=3.0,8.5 Hz), 6.91 (dd, 1H, J=5.0, 9.0 Hz), 6.62 (br s, 2H), 3.55 (s, 2H),3.19 (s, 2H), 2.59 (br s, 4H), 2.49 (br s, 4H). ¹³C-NMR (125 MHz,(CD₃)₂CO) δ 166.8, 156.4 (d, J_(C-F)=233.5 Hz), 155.4, 155.1 (d,J_(C-F)=2.8 Hz), 143.9, 143.6, 129.9, 126.7, 119.2 (d, J_(C-F)=7.6 Hz),118.7 (d, J_(C-F)=17.8 Hz), 118.6, 116.6 (d, J_(C-F)=23.9 Hz), 62.5,61.5, 54.1, 53.4. ¹⁹F-NMR (470 MHz, (CD₃)₂CO) δ −127.3. HRMS (ESI):450.1609 (M+1); calcd. for C₂₀H₂₅FN₅O₄S: 450.1611.

2-(4-benzoylpiperazin-1-yl)-N′-(5-fluoro-2-hydroxybenzylidene)acetohydrazide(21)

Synthesized according to General Procedure D: H3 (131 mg, 0.50 mmol, 1.0equiv.), 47c (70 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) yielded 21 (157 mg,81.6%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 10.89 (br s, 1H),10.50 (br s, 1H), 8.21 (s, 1H), 7.40-7.33 (m, 5H), 6.93 (dt, 1H, J=2.5,8.5 Hz), 6.84 (dd, 1H, J=4.5, 9.0 Hz), 6.74 (dd, 1H, J=2.5, 8.5 Hz),3.79 (br s, 2H), 3.48 (br s, 2H), 3.17 (s, 2H), 2.60 (br s, 2H), 2.53(br s, 2H). ¹³C-NMR (125 MHz, CDCl₃) δ 170.5, 165.8, 155.7 (d,J_(C-F)=235.8 Hz), 154.5, 149.7, 135.3, 130.1, 128.7, 127.0, 118.8 (d,J_(C-F)=23.1 Hz), 118.2 (d, J_(C-F)=7.5 Hz), 117.6 (d, J_(C-F)=7.4 Hz),116.0 (d, J_(C-F)=23.6 Hz), 60.7, 53.6 (br), 53.4 (br), 47.6 (br), 42.0(br). ¹⁹F-NMR (470 MHz, CDCl₃) δ −128.3. HRMS (ESI): 385.1674 (M+1);calcd. for C₂₀H₂₂FN₄O₃: 385.1676.

2-(4-(4-cyanobenzyl)piperazin-1-yl)-N′-(5-fluoro-2-hydroxybenzylidene)acetohydrazide(22)

Synthesized according to General Procedure D: H9 (137 mg, 0.50 mmol, 1.0equiv.), 47c (70 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) yielded 22 (169 mg,85.5%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 10.84 (br s, 1H),10.20 (br s, 1H), 8.31 (s, 1H), 7.56 (d, 2H, J=8.5 Hz), 7.41 (d, 1H,J=8.0 Hz), 6.95 (dt, 1H, J=3.0, 9.0 Hz), 6.88-6.85 (m, 2H), 3.54 (s,2H), 3.18 (s, 2H), 2.60 (br s, 4H), 2.50 (br s, 4H). ¹³C-NMR (125 MHz,CDCl₃) δ 166.2, 155.7 (d, J_(C-F)=235.9 Hz), 154.6, 149.6, 144.0, 132.2,129.5, 118.9 (d, J_(C-F)=22.6 Hz), 118.6, 118.2 (d, J_(C-F)=7.6 Hz),117.5 (d, J_(C-F)=7.5 Hz), 116.0 (d, J_(C-F)=23.8 Hz), 110.9, 62.2,61.0, 53.6, 53.0. ¹⁹F-NMR (470 MHz, CDCl₃) δ −128.4. HRMS (ESI):396.1838 (M+1); calcd. for C₂₁H₂₃FN₅O₂: 396.1836.

2-(4-(4-cyanobenzoyl)piperazin-1-yl)-N′-(5-fluoro-2-hydroxybenzylidene)acetohydrazide(23)

Synthesized according to General Procedure D: H10 (144 mg, 0.50 mmol,1.0 equiv.), 47c (70 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) yielded 23 (144 mg,70.1%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 10.79 (br s, 1H),10.16 (br s, 1H), 8.28 (s, 1H), 7.67 (d, 2H, J=8.0 Hz), 7.47 (d, 2H,J=8.5 Hz), 6.95 (dt, 1H, J=3.0, 8.0 Hz), 6.85 (dd, 1H, J=4.5, 9.0 Hz),6.79 (dd, 1H, J=3.0, 8.5 Hz), 3.82 (br s, 2H), 3.41 (br s, 2H), 3.22 (s,2H), 2.67 (br s, 2H), 2.54 (br s, 2H). ¹³C-NMR (125 MHz, CDCl₃) δ 168.4,165.5, 155.7 (d, J_(C-F)=236.1 Hz), 154.5, 149.9, 139.7, 132.6, 127.8,119.0 (d, J_(C-F)=23.1 Hz), 118.2 (d, J_(C-F)=7.6 Hz), 118.1, 117.4 (d,J_(C-F)=7.5 Hz), 116.0 (d, J_(C-F)=23.6 Hz), 113.7, 60.8, 53.4 (br),52.7 (br), 47.4 (br), 42.0 (br). ¹⁹F-NMR (470 MHz, CDCl₃) δ −128.1. HRMS(ESI): 410.1623 (M+1); calcd. for C₂₁H₂₁FN₅O₃: 410.1628.

N′-(5-fluoro-2-hydroxybenzylidene)-2-(4-(4-fluorobenzyl)piperazin-1-yl)acetohydrazide(24)

Synthesized according to General Procedure D: H11 (133 mg, 0.50 mmol,1.0 equiv.), 47c (70 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) yielded 24 (152 mg,78.2%) as a pale yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ 10.83 (br s,1H), 10.19 (br s, 1H), 8.33 (s, 1H), 7.25 (dd, 2H, J=5.5, 8.5 Hz),6.99-6.95 (m, 3H), 6.90-6.86 (m, 2H), 3.47 (s, 2H), 3.18 (s, 2H), 2.60(br s, 4H), 2.49 (br s, 4H). ¹³C-NMR (125 MHz, CDCl₃) δ 166.3, 162.1 (d,J_(C-F)=243.9 Hz), 155.8 (d, J_(C-F)=236.0 Hz), 154.7 (d, J_(C-F)=1.4Hz), 149.7 (d, J_(C-F)=2.6 Hz), 133.7 (d, J_(C-F)=3.0 Hz), 130.6 (d,J_(C-F)=7.9 Hz), 118.8 (d, J_(C-F)=23.3 Hz), 118.3 (d, J_(C-F)=7.6 Hz),117.6 (d, J_(C-F)=7.5 Hz), 116.0 (d, J_(C-F)=23.8 Hz), 115.2 (d,J_(C-F)=21.1 Hz), 62.1, 61.0, 53.8, 52.9. ¹⁹F-NMR (470 MHz, CDCl₃) δ−118.8, −128.4. HRMS (ESI): 389.1787 (M+1); calcd. for C₂₀H₂₃F₂N₄O₂:389.1789.

N′-(5-fluoro-2-hydroxybenzylidene)-2-(4-(4-fluorobenzoyl)piperazin-1-yl)acetohydrazide(25)

Synthesized according to General Procedure D: H12 (140 mg, 0.50 mmol,1.0 equiv.), 47c (70 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) yielded 25 (101 mg,50.1%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 10.81 (br s, 1H),10.25 (br s, 1H), 8.29 (s, 1H), 7.38 (dd, 2H, J=5.5, 8.5 Hz), 7.07 (t,2H, J=8.5 Hz), 6.98-6.94 (m, 1H), 6.86 (dd, 1H, J=4.0, 9.0 Hz), 6.79(dd, 1H, J=2.0, 8.0 Hz), 3.63 (br s, 4H), 3.21 (s, 2H), 2.59 (br s, 4H).¹³C-NMR (125 MHz, CDCl₃) δ 169.7, 165.7, 163.6 (d, J_(C-F)=249.4 Hz),155.8 (d, J_(C-F)=236.3 Hz), 154.6 (d, J_(C-F)=0.9 Hz), 150.0 (d,J_(C-F)=2.3 Hz), 131.3 (d, J_(C-F)=3.4 Hz), 129.5 (d, J_(C-F)=8.4 Hz),119.0 (d, J_(C-F)=23.1 Hz), 118.3 (d, J_(C-F)=7.5 Hz), 117.5 (d,J_(C-F)=7.3 Hz), 116.0 (d, J_(C-F)=24.6 Hz), 115.8 (d, J_(C-F)=21.9 Hz),60.9, 53.5, 47.7, 42.2. ¹⁹F-NMR (470 MHz, CDCl₃) δ −112.6, −128.2. HRMS(ESI): 403.1573 (M+1);_calcd. for C₂₀H₂₁F₂N₄O₃: 403.1582.

N′-(5-fluoro-2-hydroxybenzylidene)-2-(4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)acetohydrazide(26)

Synthesized according to General Procedure D: H13 (158 mg, 0.50 mmol,1.0 equiv.), 47c (70 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) yielded 26 (194 mg,88.6%) as a pale yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ 10.83 (br s,1H), 10.17 (br s, 1H), 8.34 (s, 1H), 7.56 (d, 2H, J=8.0 Hz), 7.43 (d,2H, J=8.0 Hz), 6.98 (dt, 1H, J=3.0, 8.0 Hz), 6.91-6.86 (m, 2H), 3.57 (s,2H), 3.19 (s, 2H), 2.62 (br s, 4H), 2.52 (br s, 4H). ¹³C-NMR (125 MHz,CDCl₃) δ 166.3, 155.8 (d, J_(C-F)=236.0 Hz), 154.7 (d, J_(C-F)=1.5 Hz),149.8 (d, J_(C-F)=2.4 Hz), 142.4 (d, J_(C-F)=0.8 Hz), 129.5 (q,J_(C-F)=32.1 Hz), 129.3, 125.3 (q, J_(C-F)=3.8 Hz), 124.4 (q,J_(C-F)=270.6 Hz), 118.9 (d, J_(C-F)=23.0 Hz), 118.3 (d, J_(C-F)=7.6Hz), 117.6 (d, J_(C-F)=7.5 Hz), 116.1 (d, J_(C-F)=23.6 Hz), 62.3, 61.0,53.8, 53.1. ¹⁹F-NMR (470 MHz, CDCl₃) δ −65.4, −128.4. HRMS (ESI):439.1765 (M+1);_calcd. for C₂₁H₂₃F₄N₄O₂: 439.1757.

N′-(5-fluoro-2-hydroxybenzylidene)-2-(4-(4-(trifluoromethyl)benzoyl)piperazin-1-yl)acetohydrazide(27)

Synthesized according to General Procedure D: H14 (165 mg, 0.50 mmol,1.0 equiv.), 47c (70 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) yielded 27 (173 mg,76.5%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 10.80 (br s, 1H),10.20 (br s, 1H), 8.28 (s, 1H), 7.65 (d, 2H, J=8.0 Hz), 7.48 (d, 2H,J=7.5 Hz), 6.96 (dt, 1H, J=2.5, 8.0 Hz), 6.87 (dd, 1H, J=4.5, 8.5 Hz),6.79 (dd, 1H, J=2.5, 8.0 Hz), 3.84 (br s, 2H), 3.44 (br s, 2H), 3.22 (s,2H), 2.70 (br s, 2H), 2.55 (br s, 2H). ¹³C-NMR (125 MHz, CDCl₃) δ 169.1,165.6, 155.8 (d, J_(C-F)=236.3 Hz), 154.6 (d, J_(C-F)=1.3 Hz), 150.0 (d,J_(C-F)=1.9 Hz), 138.9, 132.0 (q, J_(C-F)=32.6 Hz), 127.5, 125.8 (q,J_(C-F)=3.6 Hz), 123.7 (q, J_(C-F)=271.3 Hz), 119.0 (d, J_(C-F)=23.1Hz), 118.3 (d, J_(C-F)=7.6 Hz), 117.4 (d, J_(C-F)=7.5 Hz), 116.0 (d,J_(C-F)=23.8 Hz), 60.8, 53.5 (br), 47.5 (br), 42.0 (br). ¹⁹F-NMR (470MHz, CDCl₃) δ −66.0, −128.1. HRMS (ESI): 453.1552 (M+1); calcd. forC₂₁H₂₁F₄N₄O₃: 453.1550.

N′-(3-allyl-5-fluoro-2-hydroxybenzylidene)-2-(4-benzylpiperazin-1-yl)acetohydrazide(28)

Synthesized according to General Procedure D: H1 (124 mg, 0.50 mmol, 1.0equiv.), 47d (90 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) yielded 28 (186 mg,90.8%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.16 (s, 1H), 10.20(br s, 1H), 8.28 (s, 1H), 7.34-7.30 (m, 4H), 7.28-7.25 (m, 1H), 6.91(dd, 1H, J=3.0, 9.0 Hz), 6.74 (dd, 1H, J=3.0, 8.0 Hz), 5.98 (tdd, 1H,J=6.5, 10.0, 16.5 Hz), 5.13-5.08 (m, 2H), 3.54 (s, 2H), 3.42 (d, 2H,J=7.0 Hz), 3.20 (s, 2H), 2.63 (br s, 4H), 2.53 (br s, 4H). ¹³C-NMR (125MHz, CDCl₃) δ 166.2, 155.5 (d, J_(C-F)=235.8 Hz), 152.5 (d, J_(C-F)=1.4Hz), 149.8 (d, J_(C-F)=2.5 Hz), 137.9, 135.7, 130.2 (d, J_(C-F)=6.8 Hz),129.2, 128.4, 127.3, 119.0 (d, J_(C-F)=23.1 Hz), 116.8 (d, J_(C-F)=7.9Hz), 116.6, 113.9 (d, J_(C-F)=23.5 Hz), 62.9, 61.0, 53.8, 53.0, 33.8.¹⁹F-NMR (470 MHz, CDCl₃) δ −128.6. HRMS (ESI): 411.2191 (M+1); calcd.for C₂₃H₂₈FN₄O₂: 411.2196.

4-((4-(2-(2-(3-allyl-5-fluoro-2-hydroxybenzylidene)hydrazinyl)-2-oxoethyl)piperazin-1-yl)methyl)benzenesulfonamide(29)

Synthesized according to General Procedure D: H2 (164 mg, 0.50 mmol, 1.0equiv.), 47d (90 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) yielded 29 (214 mg,87.2%) as a white solid. ¹H-NMR (500 MHz, (CD₃)₂CO) δ 11.73 (br s, 1H),10.94 (br s, 1H), 8.46 (s, 1H), 7.85 (d, 2H, J=8.5 Hz), 7.48 (d, 2H,J=8.5 Hz), 6.96 (d, 2H, J=9.0 Hz), 6.62 (br s, 2H), 5.99 (tdd, 1H,J=6.5, 10.0, 16.5 Hz), 5.10 (qd, 1H, J=1.5, 17.0 Hz), 5.04 (qd, 2H,J=1.5, 10.0 Hz), 3.55 (s, 2H), 3.40 (d, 2H, J=7.0 Hz), 3.19 (s, 2H),2.59 (br s, 4H), 2.49 (br s, 4H)._(—) ¹³C-NMR (125 MHz, (CD₃)₂CO) δ166.8, 156.1 (d, J_(C-F)=233.6 Hz), 153.1 (d, J_(C-F)=1.3 Hz), 149.6,143.9, 143.5, 136.6, 130.5 (d, J_(C-F)=7.0 Hz), 129.8, 126.7, 118.7 (d,J_(C-F)=23.1 Hz), 118.4 (d, J_(C-F)=8.0 Hz), 116.5, 114.6 (d,J_(C-F)=23.6 Hz), 62.5, 61.5, 54.1, 53.4, 34.2. ¹⁹F-NMR (470 MHz,(CD₃)₂CO) δ −127.4. HRMS (ESI): 490.1930 (M+1); calcd. for C₂₃H₂₉FN₅O₄S:490.1924.

N′-(3-allyl-5-fluoro-2-hydroxybenzylidene)-2-(4-benzoylpiperazin-1-yl)acetohydrazide(30)

Synthesized according to General Procedure D: H3 (131 mg, 0.50 mmol, 1.0equiv.), 47d (90 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-15% MeOH/EtOAc) yielded 30 (186 mg,87.8%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.17 (s, 1H), 10.45(br s, 1H), 8.19 (s, 1H), 7.40-7.33 (m, 5H), 6.86 (dd, 1H, J=3.0, 9.0Hz), 6.60 (dd, 1H, J=3.0, 8.5 Hz), 5.91 (tdd, 1H, J=6.5, 9.5, 18.0 Hz),5.09-5.03 (m, 2H), 3.80 (br s, 2H), 3.47 (br s, 2H), 3.35 (d, 2H, J=6.5Hz), 3.19 (s, 2H), 2.56 (br s, 4H). ¹³C-NMR (125 MHz, CDCl₃) δ 170.5,165.6, 155.4 (d, J_(C-F)=235.5 Hz), 152.4 (d, J_(C-F)=1.4 Hz), 150.1 (d,J_(C-F)=1.8 Hz), 135.6, 135.3, 130.1, 130.1, 128.7, 127.0, 119.0 (d,J_(C-F)=23.0 Hz), 116.8 (d, J_(C-F)=7.9 Hz), 116.5, 113.9 (d,J_(C-F)=23.5 Hz), 60.7, 53.6 (br), 47.6 (br), 42.0 (br), 33.7. ¹⁹F-NMR(470 MHz, CDCl₃) δ −128.5._HRMS (ESI): 425.1991 (M+1); calcd. forC₂₃H₂₆FN₄O₃: 425.1989.

N′-(3-allyl-5-fluoro-2-hydroxybenzylidene)-2-(4-(4-cyanobenzyl)piperazin-1-yl)acetohydrazide(31)

Synthesized according to General Procedure D: H9 (137 mg, 0.50 mmol, 1.0equiv.), 47d (90 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-15% MeOH/EtOAc) yielded 31 (164 mg,75.3%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.12 (br s, 1H),10.16 (br s, 1H), 8.28 (s, 1H), 7.57 (d, 2H, J=8.0 Hz), 7.42 (d, 2H,J=8.0 Hz), 6.88 (dd, 1H, J=3.0, 9.0 Hz), 6.72 (dd, 1H, J=3.0, 8.0 Hz),5.94 (tdd, 1H, J=6.5, 10.0, 17.0 Hz), 5.09-5.05 (m, 2H), 3.55 (s, 2H),3.38 (d, 2H, J=7.0 Hz), 3.19 (s, 2H), 2.62 (br s, 4H), 2.51 (br s, 4H).¹³C-NMR (125 MHz, CDCl₃) δ 166.0, 155.5 (d, J_(C-F)=235.5 Hz), 152.5 (d,J_(C-F)=1.4 Hz), 149.9, 144.0, 135.7, 132.2, 130.1 (d, J_(C-F)=6.8 Hz),129.5, 119.0 (d, J_(C-F)=23.0 Hz), 119.0, 116.8 (d, J_(C-F)=7.8 Hz),116.5, 113.9 (d, J_(C-F)=23.5 Hz), 111.0, 62.3, 60.9, 53.8, 53.1, 33.8.¹⁹F-NMR (470 MHz, CDCl₃) δ −128.6. HRMS (ESI): 436.2144 (M+1); calcd.for C₂₄H₂₇FN₅O₂: 436.2149.

N′-(3-allyl-5-fluoro-2-hydroxybenzylidene)-2-(4-(4-cyanobenzoyl)piperazin-1-yl)acetohydrazide(32)

Synthesized according to General Procedure D: H10 (144 mg, 0.50 mmol,1.0 equiv.), 47d (90 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-15% MeOH/EtOAc) yielded 32 (196 mg,87.2%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.10 (br s, 1H),10.21 (br s, 1H), 8.20 (s, 1H), 7.65 (d, 2H, J=8.0 Hz), 7.45 (d, 2H,J=8.5 Hz), 6.84 (dd, 1H, J=3.0, 9.0 Hz), 6.61 (dd, 1H, J=3.0, 8.0 Hz),5.88 (tdd, 1H, J=7.0, 10.0, 16.5 Hz), 5.03-5.00 (m, 2H), 3.81 (br s,2H), 3.40 (br s, 2H), 3.31 (d, 2H, J=6.5 Hz), 3.21 (s, 2H), 2.66 (br s,2H), 2.54 (br s, 2H). ¹³C-NMR (125 MHz, CDCl₃) δ 168.3, 165.4, 155.4 (d,J_(C-F)=235.9 Hz), 152.3, 150.1, 139.7, 135.4, 132.5, 130.0 (d,J_(C-F)=6.8 Hz), 127.7, 119.1 (d, J_(C-F)=23.1 Hz), 118.0, 116.6 (d,J_(C-F)=7.9 Hz), 116.5, 113.8 (d, J_(C-F)=23.5 Hz), 113.6, 60.7, 53.3(br), 47.3 (br), 42.0 (br), 33.6. ¹⁹F-NMR (470 MHz, CDCl₃) δ −128.3.HRMS (ESI): 450.1931 (M+1);_calcd. for C₂₄H₂₅FN₅O₃: 450.1941.

N′-(3-allyl-5-fluoro-2-hydroxybenzylidene)-2-(4-(4-fluorobenzyl)piperazin-1-yl)acetohydrazide(33)

Synthesized according to General Procedure D: H11 (133 mg, 0.50 mmol,1.0 equiv.), 47d (90 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) yielded 33 (176 mg,82.0%) as a yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.15 (br s, 1H),10.22 (br s, 1H), 8.26 (s, 1H), 7.25 (dd, 2H, J=5.5, 8.5 Hz), 6.98 (t,2H, J=8.5 Hz), 6.89 (dd, 1H, J=3.0, 9.0 Hz), 6.72 (dd, 1H, J=3.0, 8.0Hz), 5.95 (tdd, 1H, J=6.5, 10.0, 17.0 Hz), 5.10-5.06 (m, 2H), 3.47 (s,2H), 3.39 (d, 2H, J=6.5 Hz), 3.18 (s, 2H), 2.61 (br s, 4H), 2.49 (br s,4H). ¹³C-NMR (125 MHz, CDCl₃) δ 166.2, 162.1 (d, J_(C-F)=243.8 Hz),155.5 (d, J_(C-F)=235.5 Hz), 152.5 (d, J_(C-F)=0.9 Hz), 149.8, 135.7,133.6 (d, J_(C-F)=3.0 Hz), 130.6 (d, J_(C-F)=7.8 Hz), 130.1 (d,J_(C-F)=6.8 Hz), 119.0 (d, J_(C-F)=23.0 Hz), 116.8 (d, J_(C-F)=7.8 Hz),116.5, 115.1 (d, J_(C-F)=21.0 Hz), 113.9 (d, J_(C-F)=23.5 Hz), 62.1,61.0, 53.7, 52.9, 33.8. ¹⁹F-NMR (470 MHz, CDCl₃) δ −118.8, −128.6. HRMS(ESI): 429.2095 (M+1); calcd. for C₂₃H₂₇F₂N₄O₂: 429.2102.

N′-(3-allyl-5-fluoro-2-hydroxybenzylidene)-2-(4-(4-fluorobenzoyl)piperazin-1-yl)acetohydrazide(34)

Synthesized according to General Procedure D: H12 (140 mg, 0.50 mmol,1.0 equiv.), 47d (90 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-15% MeOH/EtOAc) yielded 34 (163 mg,73.8%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.10 (br s, 1H),10.23 (br s, 1H), 8.25 (s, 1H), 7.38 (dd, 2H, J=5.5, 8.5 Hz), 7.07 (t,2H, J=8.5 Hz), 6.88 (dd, 1H, J=3.0, 9.0 Hz), 6.65 (dd, 1H, J=3.0, 8.5Hz), 5.92 (tdd, 1H, J=6.5, 9.5, 17.0 Hz), 5.10-5.04 (m, 2H), 3.82 (br s,2H), 3.50 (br s, 2H), 3.36 (d, 2H, J=6.5 Hz), 3.21 (s, 2H), 2.59 (br s,4H). ¹³C-NMR (125 MHz, CDCl₃) δ 169.7, 165.6, 163.6 (d, J_(C-F)=249.8Hz), 155.5 (d, J_(C-F)=235.6 Hz), 152.5 (d, J_(C-F)=1.4 Hz), 150.3 (d,J_(C-F)=2.5 Hz), 135.6, 131.3 (d, J_(C-F)=3.5 Hz), 130.2 (d, J_(C-F)=6.9Hz), 129.5 (d, J_(C-F)=8.4 Hz), 119.2 (d, J_(C-F)=23.1 Hz), 116.7 (d,J_(C-F)=7.8 Hz), 116.6, 115.9 (d, J_(C-F)=21.8 Hz), 113.9 (d,J_(C-F)=23.5 Hz), 60.9, 53.6 (br), 47.7 (br), 42.2 (br), 33.8. ¹⁹F-NMR(470 MHz, CDCl₃) δ −112.6, −128.4. HRMS (ESI): 443.1886 (M+1); calcd.for C₂₃H₂₅F₂N₄O₃: 443.1895.

N′-(3-allyl-5-fluoro-2-hydroxybenzylidene)-2-(4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)acetohydrazide(35)

Synthesized according to General Procedure D: H13 (158 mg, 0.50 mmol,1.0 equiv.), 47d (90 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) yielded 35 (176 mg,73.7%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.14 (br s, 1H),10.19 (br s, 1H), 8.28 (s, 1H), 7.56 (d, 2H, J=8.0 Hz), 7.43 (d, 2H,J=8.0 Hz), 6.90 (dd, 1H, J=3.0, 9.0 Hz), 6.72 (dd, 2H, J=3.0, 8.0 Hz),5.96 (tdd, 1H, J=6.5, 10.0, 17.0 Hz), 5.11-5.08 (m, 2H), 3.57 (s, 2H),3.40 (d, 2H, J=6.5 Hz), 3.20 (s, 2H), 2.63 (br s, 4H), 2.53 (br s, 4H).¹³C-NMR (125 MHz, CDCl₃) δ 166.1, 155.6 (d, J_(C-F)=235.5 Hz), 152.5 (d,J_(C-F)=1.3 Hz), 149.9 (d, J_(C-F)=2.4 Hz), 142.4, 135.7, 130.2 (d,J_(C-F)=6.8 Hz), 129.5 (q, J_(C-F)=32.0 Hz), 129.3, 125.3 (q,J_(C-F)=3.8 Hz), 124.4 (q, J_(C-F)=270.5 Hz), 119.1 (d, J_(C-F)=23.1Hz), 116.9 (d, J_(C-F)=7.8 Hz), 116.6, 114.0 (d, J_(C-F)=23.5 Hz), 62.3,61.0, 53.8, 53.1, 33.8. ¹⁹F-NMR (470 MHz, CDCl₃) δ −65.4, −128.5. HRMS(ESI): 479.2066 (M+1); calcd. for C₂₄H₂₇F₄N₄O₂: 479.2070.

N′-(3-allyl-5-fluoro-2-hydroxybenzylidene)-2-(4-(4-(trifluoromethyl)benzoyl)piperazin-1-yl)acetohydrazide(36)

Synthesized according to General Procedure D: H14 (165 mg, 0.50 mmol,1.0 equiv.), 47d (90 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-15% MeOH/EtOAc) yielded 36 (139 mg,56.3%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.07 (br s, 1H),10.14 (br s, 1H), 8.26 (s, 1H), 7.66 (d, 2H, J=8.5 Hz), 7.50 (d, 2H,J=8.0 Hz), 6.89 (dd, 1H, J=3.0, 9.0 Hz), 6.66 (dd, 1H, J=3.0, 8.5 Hz),5.93 (tdd, 1H, J=7.0, 10.0, 16.5 Hz), 5.10-5.04 (m, 2H), 3.86 (br s,2H), 3.45 (br s, 2H), 3.37 (d, 2H, J=6.5 Hz), 3.23 (s, 2H), 2.69 (br s,2H), 2.57 (br s, 2H). ¹³C-NMR (125 MHz, CDCl₃) δ 169.1, 165.5, 155.6 (d,J_(C-F)=235.9 Hz), 152.5, 150.4 (d, J_(C-F)=2.1 Hz), 138.9, 135.6, 132.0(q, J_(C-F)=32.6 Hz), 130.3 (d, J_(C-F)=6.8 Hz), 127.5, 125.9 (q,J_(C-F)=3.8 Hz), 123.7 (q, J_(C-F)=271.1 Hz), 119.3 (d, J_(C-F)=23.0Hz), 116.7 (d, J_(C-F)=4.6 Hz), 116.7, 114.0 (d, J_(C-F)=23.5 Hz), 60.9,53.6 (br), 47.5 (br), 42.2 (br), 33.8. ¹⁹F-NMR (470 MHz, CDCl₃) δ −66.0,−128.4. HRMS (ESI): 493.1868 (M+1);_calcd. for C₂₄H₂₅F₄N₄O₃: 493.1863.

2-(4-benzylpiperazin-1-yl)-N′-(5-fluoro-2-hydroxy-3-propylbenzylidene)acetohydrazide(37)

Synthesized according to General Procedure D: H1 (124 mg, 0.50 mmol, 1.0equiv.), 47e (91 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-15% MeOH/EtOAc) yielded 37 (174 mg,84.6%) as an off-white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.10 (s, 1H),10.19 (br s, 1H), 8.26 (s, 1H), 7.32-7.30 (m, 4H), 7.28-7.25 (m, 1H),6.89 (dd, 1H, J=3.0, 9.0 Hz), 6.71 (dd, 1H, J=3.0, 8.5 Hz), 3.54 (s,2H), 3.20 (s, 2H), 2.66-2.59 (m, 6H), 2.53 (br s, 4H), 1.64 (sext, 2H,J=7.5 Hz), 0.95 (t, 3H, J=7.5 Hz). ¹³C-NMR (125 MHz, CDCl₃) δ 166.1,155.4 (d, J_(C-F)=235.1 Hz), 152.8, 150.0, 137.9, 132.7 (d, J_(C-F)=6.6Hz), 129.2, 128.4, 127.3, 119.1 (d, J_(C-F)=22.5 Hz), 116.7 (d,J_(C-F)=7.9 Hz), 113.4 (d, J_(C-F)=23.4 Hz), 63.0, 61.0, 53.8, 53.1,31.9, 22.5, 14.0. ¹⁹F-NMR (470 MHz, CDCl₃) δ −129.1. HRMS (ESI):413.2345 (M+1); calcd. for C₂₃H₃₀FN₄O₂: 413.2353.

4-((4-(2-(2-(5-fluoro-2-hydroxy-3-propylbenzylidene)hydrazinyl)-2-oxoethyl)piperazin-1-yl)methyl)benzenesulfonamide(38)

Synthesized according to General Procedure D: H2 (164 mg, 0.50 mmol, 1.0equiv.), 47e (91 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) yielded 38 (221 mg,89.9%) as a white solid. ¹H-NMR (500 MHz, (CD₃)₂CO) δ 11.68 (br s, 1H),10.92 (br s, 1H), 8.46 (s, 1H), 7.84 (d, 2H, J=8.0 Hz), 7.49 (d, 2H,J=8.0 Hz), 6.98 (dd, 1H, J=3.0, 9.5 Hz), 6.93 (dd, 1H, J=3.0, 8.5 Hz),6.59 (br s, 2H), 3.57 (s, 2H), 3.18 (s, 2H), 2.64-2.59 (m, 6H), 2.50 (brs, 4H), 1.63 (sext, 2H, J=7.5 Hz), 0.93 (t, 3H, J=7.5 Hz). ¹³C-NMR (125MHz, (CD₃)₂CO) δ 166.6, 156.1 (d, J_(C-F)=233.1 Hz), 153.4 (d,J_(C-F)=1.4 Hz), 149.7 (d, J_(C-F)=2.9 Hz), 143.9, 143.6, 132.8 (d,J_(C-F)=6.9 Hz), 129.9, 126.8, 119.0 (d, J_(C-F)=22.8 Hz), 118.3 (d,J_(C-F)=8.1 Hz), 114.2 (d, J_(C-F)=23.6 Hz), 62.5, 61.6, 54.2, 53.4,32.3, 23.1, 14.1. ¹⁹F-NMR (470 MHz, (CD₃)₂CO) δ −127.7. HRMS (ESI):492.2074 (M+1); calcd. for C₂₃H₃₁FN₅O₄S: 492.2081.

2-(4-benzoylpiperazin-1-yl)-N′-(5-fluoro-2-hydroxy-3-propylbenzylidene)acetohydrazide(39)

Synthesized according to General Procedure D: H3 (131 mg, 0.50 mmol, 1.0equiv.), 47e (91 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-10% MeOH/EtOAc) yielded 39 (189 mg,88.9%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.13 (s, 1H), 10.48(br s, 1H), 8.15 (s, 1H), 7.38-7.32 (m, 5H), 6.83 (dd, 1H, J=3.0, 9.0Hz), 6.55 (dd, 1H, J=3.0, 8.5 Hz), 3.80 (br s, 2H), 3.46 (br s, 2H),3.18 (s, 2H), 2.59-2.54 (m, 6H), 1.57 (sext, 2H, J=7.5 Hz), 0.88 (t, 3H,J=7.5 Hz). ¹³C-NMR (125 MHz, CDCl₃) δ 170.5, 165.6, 155.3 (d,J_(C-F)=235.1 Hz), 152.6 (d, J_(C-F)=1.4 Hz), 150.2 (d, J_(C-F)=2.5 Hz),135.3, 132.5 (d, J_(C-F)=6.8 Hz), 130.1, 128.7, 126.9, 119.1 (d,J_(C-F)=22.6 Hz), 116.6 (d, J_(C-F)=7.9 Hz), 113.4 (d, J_(C-F)=23.4 Hz),60.7, 53.5 (br), 47.5 (br), 42.0 (br), 31.8, 22.4, 13.9. ¹⁹F-NMR (470MHz, CDCl₃) δ −129.0. HRMS (ESI): 427.2144 (M+1); calcd. forC₂₃H₂₈FN₄O₃: 427.2145.

2-(4-(4-cyanobenzyl)piperazin-1-yl)-N′-(5-fluoro-2-hydroxy-3-propylbenzylidene)acetohydrazide(40)

Synthesized according to General Procedure D: H9 (137 mg, 0.50 mmol, 1.0equiv.), 47e (91 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-15% MeOH/EtOAc) yielded 40 (180 mg,82.1%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.07 (br s, 1H),10.16 (br s, 1H), 8.25 (s, 1H), 7.57 (d, 2H, J=8.5 Hz), 7.42 (d, 2H,J=8.0 Hz), 6.86 (dd, 1H, J=3.0, 9.0 Hz), 6.68 (dd, 1H, J=3.0, 8.0 Hz),3.55 (s, 2H), 3.18 (s, 2H), 2.70-2.57 (m, 6H), 2.51 (br s, 4H). ¹³C-NMR(125 MHz, CDCl₃) δ 166.0, 155.4 (d, J_(C-F)=235.1 Hz), 152.7 (d,J_(C-F)=1.4 Hz), 150.0 (d, J_(C-F)=2.5 Hz), 144.0, 132.6 (d, J_(C-F)=6.8Hz), 132.2, 129.5, 119.1 (d, J_(C-F)=22.6 Hz), 119.0, 116.6 (d,J_(C-F)=8.0 Hz), 113.4 (d, J_(C-F)=23.5 Hz), 110.9, 62.3, 60.9, 53.7,53.1, 31.9, 22.4, 14.0. ¹⁹F-NMR (470 MHz, CDCl₃) δ −129.0. HRMS (ESI):438.2301 (M+1);_calcd. for C₂₄H₂₉FN₅O₂: 438.2305.

2-(4-(4-cyanobenzoyl)piperazin-1-yl)-N′-(5-fluoro-2-hydroxy-3-propylbenzylidene)acetohydrazide(41)

Synthesized according to General Procedure D: H10 (144 mg, 0.50 mmol,1.0 equiv.), 47e (91 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-10% MeOH/EtOAc) yielded_(—)41 (196mg, 86.5%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.03 (br s, 1H),10.17 (br s, 1H), 8.19 (s, 1H), 7.66 (d, 2H, J=8.0 Hz), 7.46 (d, 2H,J=8.0 Hz), 6.84 (dd, 1H, J=3.0, 9.0 Hz), 6.59 (dd, 1H, J=3.0, 8.5 Hz),3.82 (br s, 2H), 3.41 (br s, 2H), 3.22 (s, 2H), 2.66 (br s, 2H),2.57-2.52 (m, 2H), 1.55 (sext, 2H, J=7.5 Hz), 0.87 (t, 3H, J=7.5 Hz).¹³C-NMR (125 MHz, CDCl₃) δ 168.3, 165.4, 155.3 (d, J_(C-F)=235.5 Hz),152.6 (d, J_(C-F)=1.1 Hz), 150.3 (d, J_(C-F)=2.0 Hz), 139.7, 132.6 (d,J_(C-F)=6.8 Hz), 132.5, 127.7, 119.2 (d, J_(C-F)=22.6 Hz), 118.0, 116.4(d, J_(C-F)=7.9 Hz), 113.6, 113.4 (d, J_(C-F)=23.3 Hz), 60.7, 53.4 (br),47.4 (br), 42.0 (br), 31.7, 22.3, 13.9. ¹⁹F-NMR (470 MHz, CDCl₃) δ−128.7. HRMS (ESI): 452.2098 (M+1);_calcd. for C₂₄H₂₇FN₅O₃: 452.2098.

N′-(5-fluoro-2-hydroxy-3-propylbenzylidene)-2-(4-(4-fluorobenzyl)piperazin-1-yl)acetohydrazide(42)

Synthesized according to General Procedure D: H11 (133 mg, 0.50 mmol,1.0 equiv.), 47e (91 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) yielded 42 (202 mg,93.8%) as a yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.07 (br s, 1H),10.16 (br s, 1H), 8.26 (s, 1H), 7.26 (dd, 2H, J=5.5, 8.5 Hz), 6.99 (t,2H, J=8.5 Hz), 6.88 (dd, 1H, J=3.0, 9.0 Hz), 6.70 (dd, 1H, J=3.0, 8.0Hz), 3.48 (s, 2H), 3.18 (s, 2H), 2.66-2.58 (m, 6H), 2.50 (br s, 4H),1.62 (sext, 2H, J=7.5 Hz), 0.94 (t, 3H, J=7.5 Hz). ¹³C-NMR (125 MHz,CDCl₃) δ 166.1, 162.1 (d, J_(C-F)=243.8 Hz), 155.5 (d, J_(C-F)=235.0Hz), 152.8 (d, J_(C-F)=0.9 Hz), 150.1 (d, J_(C-F)=2.0 Hz), 133.7, 132.7(d, J_(C-F)=6.8 Hz), 130.7 (d, J_(C-F)=7.9 Hz), 119.2 (d, J_(C-F)=22.5Hz), 116.7 (d, J_(C-F)=7.9 Hz), 115.2 (d, J_(C-F)=21.0 Hz), 113.5 (d,J_(C-F)=23.4 Hz), 62.1, 61.0, 53.8, 53.0, 31.9, 22.5, 14.1. ¹⁹F-NMR (470MHz, CDCl₃) δ −118.8, −129.0. HRMS (ESI): 431.2250 (M+1); calcd. forC₂₃H₂₉F₂N₄O₂: 431.2259.

N′-(5-fluoro-2-hydroxy-3-propylbenzylidene)-2-(4-(4-fluorobenzoyl)piperazin-1-yl)acetohydrazide(43)

Synthesized according to General Procedure D: H12 (140 mg, 0.50 mmol,1.0 equiv.), 47e (91 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-10% MeOH/EtOAc) yielded 43 (195 mg,87.7%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.07 (br s, 1H),10.32 (br s, 1H), 8.18 (s, 1H), 7.36 (dd, 2H, J=5.0, 8.5 Hz), 7.05 (t,2H, J=8.5 Hz), 6.84 (dd, 1H, J=3.0, 9.0 Hz), 6.57 (dd, 1H, J=3.0, 8.5Hz), 3.81 (br s, 2H), 3.48 (br s, 2H), 3.20 (s, 2H), 2.62-2.50 (m, 6H),1.56 (sext, 2H, J=7.5 Hz), 0.88 (t, 3H, J=7.5 Hz). ¹³C-NMR (125 MHz,CDCl₃) δ 169.6, 165.6, 163.5 (d, J_(C-F)=249.3 Hz), 155.4 (d,J_(C-F)=235.4 Hz), 152.7 (d, J_(C-F)=0.9 Hz), 150.3, 132.6 (d,J_(C-F)=6.8 Hz), 131.3 (d, J_(C-F)=3.4 Hz), 129.4 (d, J_(C-F)=8.4 Hz),119.2 (d, J_(C-F)=22.6 Hz), 116.5 (d, J_(C-F)=7.9 Hz), 115.8 (d,J_(C-F)=21.6 Hz), 113.4 (d, J_(C-F)=23.4 Hz), 60.7, 53.5 (br), 47.7(br), 42.1 (br), 31.8, 22.4, 13.9. ¹⁹F-NMR (470 MHz, CDCl₃) δ −112.6,−128.8. HRMS (ESI): 445.2049 (M+1); calcd. for C₂₃H₂₇F₂N₄O₃: 445.2051.

N′-(5-fluoro-2-hydroxy-3-propylbenzylidene)-2-(4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)acetohydrazide(44)

Synthesized according to General Procedure D: H13 (158 mg, 0.50 mmol,1.0 equiv.), 47e (91 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-20% MeOH/EtOAc) yielded 44 (184 mg,76.5%) as a light yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.08 (br s,1H), 10.18 (br s, 1H), 8.25 (s, 1H), 7.56 (d, 2H, J=8.0 Hz), 7.43 (d,2H, J=8.0 Hz), 6.88 (dd, 1H, J=3.0, 9.0 Hz), 6.69 (dd, 2H, J=3.0, 8.5Hz), 3.56 (s, 2H), 3.20 (s, 2H), 2.64-2.58 (m, 6H), 2.52 (br s, 4H),1.62 (sext, 2H, J=7.5 Hz), 0.93 (t, 3H, J=7.5 Hz). ¹³C-NMR (125 MHz,CDCl₃) δ 166.1, 155.5 (d, J_(C-F)=235.1 Hz), 152.8 (d, J_(C-F)=1.2 Hz),150.0 (d, J_(C-F)=2.6 Hz), 142.4, 132.7 (d, J_(C-F)=6.8 Hz), 129.5 (q,J_(C-F)=32.1 Hz), 129.3, 125.3 (q, J_(C-F)=3.8 Hz), 124.4 (q,J_(C-F)=270.5 Hz), 119.2 (d, J_(C-F)=22.6 Hz), 116.7 (d, J_(C-F)=7.9Hz), 113.5 (d, J_(C-F)=23.5 Hz), 62.3, 61.0, 53.8, 53.1, 31.9, 22.5,14.0. ¹⁹F-NMR (470 MHz, CDCl₃) δ −65.4, −128.9. HRMS (ESI): 481.2216(M+1); calcd. for C₂₄H₂₉F₄N₄O₂: 481.2227.

N′-(5-fluoro-2-hydroxy-3-propylbenzylidene)-2-(4-(4-(trifluoromethyl)benzoyl)piperazin-1-yl)acetohydrazide(45)

Synthesized according to General Procedure D: H14 (165 mg, 0.50 mmol,1.0 equiv.), 47e (91 mg, 0.50 mmol, 1.0 equiv.), 1.2 M HCl (29 μL, 0.035mmol, 0.070 equiv.), EtOH (3 mL, 0.15 M). Purification by silica gelcolumn chromatography (gradient, 0-10% MeOH/EtOAc) yielded 45 (140 mg,56.7%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 11.04 (br s, 1H),10.19 (br s, 1H), 8.21 (s, 1H), 7.65 (d, 2H, J=8.5 Hz), 7.48 (d, 2H,J=8.0 Hz), 6.86 (dd, 1H, J=3.0, 9.0 Hz), 6.60 (dd, 1H, J=3.0, 8.0 Hz),3.85 (br s, 2H), 3.44 (br s, 2H), 3.22 (s, 2H), 2.67 (br s, 2H),2.60-2.51 (m, 4H), 1.58 (sext, 2H, J=7.5 Hz), 0.90 (t, 3H, J=7.5 Hz).¹³C-NMR (125 MHz, CDCl₃) δ 169.0, 165.5, 155.4 (d, J_(C-F)=235.4 Hz),152.7 (d, J_(C-F)=0.9 Hz), 150.4 (d, J_(C-F)=2.4 Hz), 138.9, 132.7 (d,J_(C-F)=6.8 Hz), 132.0 (q, J_(C-F)=32.6 Hz), 127.5, 125.8 (q,J_(C-F)=3.6 Hz), 123.7 (q, J_(C-F)=271.0 Hz), 119.3 (d, J_(C-F)=22.6Hz), 116.5 (d, J_(C-F)=7.9 Hz), 113.4 (d, J_(C-F)=23.4 Hz), 60.8, 53.5(br), 47.5 (br), 42.0 (br), 31.8, 22.4, 14.0. ¹⁹F-NMR (470 MHz, CDCl₃) δ−66.0, −128.7. HRMS (ESI): 495.2008 (M+1); calcd. for C₂₄H₂₇F₄N₄O₃:495.2019.

Example 3 CITATIONS

-   1. Putt, K. S.; Chen, G. W.; Pearson, J. M.; Sandhorst, J. S.;    Hoagland, M. S.; Kwon, J. T.; Hwang, S. K.; Jin, H.; Churchwell, M.    I.; Cho, M. H.; Doerge, D. R.; Helferich, W. G.; Hergenrother, P.    J., Small-molecule activation of procaspase-3 to caspase-3 as a    personalized anticancer strategy. Nat. Chem. Biol. 2006, 2, 543-550.-   2. Peterson, Q. P.; Hsu, D. C.; Goode, D. R.; Novotny, C. J.;    Totten, R. K.; Hergenrother, P. J., Procaspase-3 Activation as an    Anti-Cancer Strategy: Structure-Activity Relationship of    Procaspase-Activating Compound 1 (PAC-1) and Its Cellular    Co-Localization with Caspase-3. J. Med. Chem. 2009, 52, 5721-5731.-   3. Hsu, D. C.; Roth, H. S.; West, D. C.; Botham, R. C.; Novotny, C.    J.; Schmid, S. C.; Hergenrother, P. J., Parallel Synthesis and    Biological Evaluation of 837 Analogues of Procaspase-Activating    Compound 1 (PAC-1). ACS Comb. Sci. 2012, 14, 44-50.-   4. Vichai, V.; Kirtikara, K., Sulforhodamine B colorimetric assay    for cytotoxicity screening. Nat. Protoc. 2006, 1, 1112-1116.

Example 5 Pharmaceutical Dosage Forms

The following formulations illustrate representative pharmaceuticaldosage forms that may be used for the therapeutic or prophylacticadministration of a compound of a formula described herein, a compoundspecifically disclosed herein, or a pharmaceutically acceptable salt orsolvate thereof (hereinafter referred to as ‘Compound X’):

(i) Tablet 1 mg/tablet ‘Compound X’ 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0

(ii) Tablet 2 mg/tablet ‘Compound X’ 20.0 Microcrystalline cellulose410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0500.0

(iii) Capsule mg/capsule ‘Compound X’ 10.0 Colloidal silicon dioxide 1.5Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0

(iv) Injection 1 (1 mg/mL) mg/mL ‘Compound X’ (free acid form) 1.0Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodiumchloride 4.5 1.0N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(v) Injection 2 (10 mg/mL) mg/mL ‘Compound X’ (free acid form) 10.0Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethyleneglycol 400 200.0 0.1N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(vi) Aerosol mg/can ‘Compound X’ 20 Oleic acid 10Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000Dichlorotetrafluoroethane 5,000

(vii) Topical Gel 1 wt. % ‘Compound X’   5% Carbomer 934 1.25%Triethanolamine q.s. (pH adjustment to 5-7) Methyl paraben  0.2%Purified water q.s. to 100 g

(viii) Topical Gel 2 wt. % ‘Compound X’ 5% Methylcellulose 2% Methylparaben 0.2%  Propyl paraben 0.02%   Purified water q.s. to 100 g

(ix) Topical Ointment wt. % ‘Compound X’ 5% Propylene glycol 1%Anhydrous ointment base 40%  Polysorbate 80 2% Methyl paraben 0.2% Purified water q.s. to 100 g

(x) Topical Cream 1 wt. % ‘Compound X’  5% White bees wax 10% Liquidparaffin 30% Benzyl alcohol  5% Purified water q.s. to 100 g

(xi) Topical Cream 2 wt. % ‘Compound X’ 5% Stearic acid 10%  Glycerylmonostearate 3% Polyoxyethylene stearyl ether 3% Sorbitol 5% Isopropylpalmitate 2% Methyl Paraben 0.2%  Purified water q.s. to 100 g

These formulations may be prepared by conventional procedures well knownin the pharmaceutical art. It will be appreciated that the abovepharmaceutical compositions may be varied according to well-knownpharmaceutical techniques to accommodate differing amounts and types ofactive ingredient ‘Compound X’. Aerosol formulation (vi) may be used inconjunction with a standard, metered dose aerosol dispenser.Additionally, the specific ingredients and proportions are forillustrative purposes. Ingredients may be exchanged for suitableequivalents and proportions may be varied, according to the desiredproperties of the dosage form of interest.

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, such embodiments are onlyillustrative and do not limit the scope of the invention. Changes andmodifications can be made in accordance with ordinary skill in the artwithout departing from the invention in its broader aspects as definedin the following claims.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Nolimitations inconsistent with this disclosure are to be understoodtherefrom. The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

1. A compound that directly activates procaspase-3 upon contactingprocaspase-3, wherein the compound is a novel compound of Formula I:

wherein R¹ is an optionally substituted benzoyl; n is 1, 2, 3, or 4; andeach R² is independently H, alkyl, alkoxy, hydroxy, carboxy, halo,amino, alkylamino, dialkylamino, trifluoromethyl, trifluoromethoxy,benzyl, benzyloxy, nitro, cyano (—CN), sulfonamide (—SO₂NH₂),2-propenyl, acetylene, N-alkyl-triazole, or N-benzyl-triazole; or two R²groups form an ortho-fused benzo group; or a pharmaceutically acceptablesalt or solvate thereof.
 2. The compound of claim 1 wherein n is 1 or 2.3. The compound of claim 1 wherein R² is methyl, t-butyl, methoxy,hydroxy, fluoro, chloro, bromo, iodo, amino, ethylamino, diethylamino,trifluoromethoxy, benzyl, benzyloxy, nitro, 2-propenyl, acetylene,N-methyl-triazole, or N-benzyl-triazole.
 4. The compound of claim 1wherein n is 2 and two R² groups form an ortho-fused benzo group.
 5. Thecompound of claim 1 wherein n is 2 and each R² is t-butyl.
 6. Thecompound of claim 1 wherein n is 1 and R² is 2-propenyl.
 7. The compoundof claim 1 wherein R¹ is a methoxy-benzoyl; dimethoxy-benzoyl;benzyloxy-benzoyl; t-butyl-benzoyl; naphthylcarbonyl; or ethyl-benzoyl.8. The compound of claim 1 wherein R¹ is 4-methoxy-benzoyl;2,5-dimethoxy-benzoyl; 4-benzyloxy-benzoyl; 4-t-butyl-benzoyl;2-naphthylcarbonyl; or 4-ethyl-benzoyl.
 9. The compound of claim 1wherein R¹ is:
 10. The compound of claim 1 wherein the compound is:


11. The compound of claim 1 wherein the compound induces death of cancercells in culture.
 12. A pharmaceutical composition comprising a compoundof claim 1 and a pharmaceutically acceptable diluent, excipient, orcarrier.
 13. A method of treating a cancer cell comprising (a)identifying a susceptibility to treatment of a cancer cell with aprocaspase activator compound; and (b) exposing a cancer cell to aneffective amount of the procaspase activator compound; wherein theprocaspase activator compound is a compound of claim
 1. 14. A method ofinducing apoptosis in a cancer cell comprising administering to a cancercell an effective amount of a compound of claim
 1. 15. A compound ofclaim 1 wherein the compound is a compound of Formula (X):

wherein R¹⁰ is H, F, Cl, Br, —NO₂, —CN, —CF₃, —OCF₃, or —SO₂NH₂; R²⁰ isH, F, Cl, Br, —NO₂, —CN, —CF₃, —OCF₃, or —SO₂NH₂; and R³⁰ is H,(C₁-C₆)alkyl, (C₁-C₆)alkenyl, or (C₁-C₆)alkoxy; or a pharmaceuticallyacceptable salt or solvate thereof.
 16. The compound of claim 15 whereinR³⁰ is H, propyl, or 2-propenyl.
 17. A pharmaceutical compositioncomprising a compound of claim 15 and a pharmaceutically acceptablediluent, excipient, or carrier.
 18. A method of inducing apoptosis in acancer cell comprising administering to a cancer cell an effectiveamount of a compound of claim
 15. 19. The method of claim 18 wherein thecancer cell is an adrenal cancer cell, a brain cancer cell, a breastcancer cell, a colon cancer cell, a kidney cancer cell, a lymphoma cell,a leukemia cell, a liver cancer cell, a lung cancer cell, a melanomacell, a neuroblastoma cell, a pancreatic cancer cell, a prostate cancercell, or a renal cancer cell.
 20. The method of claim 14 wherein thecancer cell is an adrenal cancer cell, a brain cancer cell, a breastcancer cell, a colon cancer cell, a kidney cancer cell, a lymphoma cell,a leukemia cell, a liver cancer cell, a lung cancer cell, a melanomacell, a neuroblastoma cell, a pancreatic cancer cell, a prostate cancercell, or a renal cancer cell.